Methods for regulating inflammatory mediators and peptides for useful therein

ABSTRACT

The present invention includes methods of modulating cellular secretory processes. More specifically the present invention relates to modulating or reducing the release of inflammatory mediators from inflammatory cells by inhibiting the mechanism associated with the release of inflammatory mediators from the vesicles or granules in the inflammatory cells in a subject with a chronic inflammatory disease. In this regard, the present invention discloses an intracellular signaling mechanism that illustrates several novel intracellular targets for pharmacological intervention in disorders involving secretion of inflammatory mediators from vesicles in inflammatory cells. MANS peptide and active fragments thereof are useful in such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/367,449, filed on Mar. 6, 2006, now U.S. Pat.No. 7,544,772, which is a continuation-in-part application of U.S.patent application Ser. No. 10/802,644, filed on Mar. 17, 2004, nowabandoned, which is a continuation application of U.S. patentapplication Ser. No. 10/180,753; filed Jun. 26, 2002, now abandoned,which claims priority to U.S. Provisional Application No. 60/300,933,filed Jun. 26, 2001, the disclosures of which are all incorporatedherein by reference in their entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with support from the United States Federalgovernment under grant number R01 HL36982 from the National Institutesof Health. The United States government may have certain rights in thisinvention.

FIELD OF INVENTION

The present invention relates to methods of modulating cellularsecretory processes. More specifically the present invention relates tomodulating the release of inflammatory mediators. The present inventionalso relates to the intracellular signaling mechanism regulating thesecretion of inflammatory mediators from membrane-bound vesicles orgranules in inflammatory cells.

BACKGROUND OF THE INVENTION

Hypersecretion of mucus contributes to the pathogenesis of a largenumber of airway inflammatory diseases in both human and non-humananimals. Increased mucus secretion is seen in chronic disease statessuch as asthma, COPD and chronic bronchitis; in genetic diseases such ascystic fibrosis; in allergic conditions (atopy, allergic inflammation);in bronchiectasis; and in a number of acute, infectious respiratoryillnesses such as pneumonia, rhinitis, influenza or the common cold.

Accompanying hypersecretion of mucus in many of these respiratorydiseases is the constant presence of inflammatory cells in the airways.These cells contribute greatly to the pathology of these diseases viathe tissue damage done by the inflammatory mediators released from thesecells. One example of such destruction via this chronic inflammationoccurs in cystic fibrosis patients where mediators released fromneutrophils (e.g., myeloperoxidase) induce the desquamation of theairway epithelial tissue.

Under-secretion of mucus also has harmful effects. Airway mucus acts asa physical barrier against biologically active inhaled particles, andmay help prevent bacterial colonization of the airways and inactivatecytotoxic products released from leukocytes. King et al., Respir.Physiol. 62:47-59 (1985); Vishwanath and Ramphal, Infect. Immun. 45:197(1984); Cross et al., Lancet 1:1328 (1984). In the eye, mucus maintainsthe tear film, and is important for eye health and comfort. Mucussecretion in the gastrointestinal tract also has a cytoprotectivefunction. The role of mucus as a chemical, biological and mechanicalbarrier means that abnormally low mucus secretion by mucous membranes isundesirable.

Mammalian airways are lined by a thin layer of mucus produced andsecreted by airway epithelial (goblet) cells and submucosal glands. Inairway diseases such as asthma, chronic bronchitis, and cystic fibrosis,hypersecretion of mucus is a common symptom. Excess mucus can contributeto obstruction and susceptibility to infection. The major components ofmucus are mucin glycoproteins synthesized by secretory cells and storedwithin cytoplasmic granules. Mucins are a family of glycoproteinssecreted by the epithelial cells including those at the respiratory,gastrointestinal and female reproductive tracts. Mucins are responsiblefor the viscoelastic properties of mucus and at least eight mucin genesare known. Thornton, et al., J. Biol. Chem. 272, 9561-9566 (1997).Mucociliary impairment caused by mucin hypersecretion and/or mucus cellhyperplasia leads to airway mucus plugging that promotes chronicinfection, airflow obstruction and sometimes death. Many airwaydiseases, such as chronic bronchitis, chronic obstructive pulmonarydisease, bronchiectacis, asthma, cystic fibrosis and bacterialinfections are characterized by mucin overproduction. E. Prescott, etal., Eur. Respir. J., 8:1333-1338 (1995); K. C. Kim, et al., Eur.Respir. J., 10:1438 (1997); D. Steiger, et al. Am. J. Respir. Cell Mol.Biol., 12:307-314 (1995). Upon appropriate stimulation, mucin granulesare released via an exocytotic process in which the granules translocateto the cell periphery where the granule membranes fuse with the plasmamembrane, allowing for luminal secretion of the contents.

Despite the obvious pathophysiological importance of this process,intracellular signaling mechanisms linking stimulation at the cellsurface to mucin granule release has only recently been elucidated. See,Li et al., Journal of Biological Chemistry, 276: 40982-40990 (2001). Itis known that a wide variety of agents and inflammatory/humoralmediators provoke mucin secretion. These include cholinergic agonists,lipid mediators, oxidants, cytokines, neuropeptides, ATP and UTP,bacterial products, neutrophil elastase, and inhaled pollutants. See,Adler et al., Res. Immunol. 149, 245-248 (1998). Interestingly, many ofthese mucin secretagogues are also known to activate several proteinkinases, and studies examining the regulation of excess secretion ofmucin by airway epithelial cells from various species have consistentlyimplicated involvement of either protein kinase C (PKC) orcGMP-dependent protein kinase (PKG) in the secretory process. See, e.g.,Ko et al., Am. J. Respir. Cell Mol. Biol. 16, 194-198 (1997); Abdullahet al., Am. J. Physiol. 273, L201-L210 (1997); Abdullah et al., Biochem.J. 316, 943-954 (1996); Larivee et al. Am. J. Respir. Cell Mol. Biol.11, 199-205 (1994); and Fischer et al., Am. J. Respir. Cell Mol. Biol.20, 413-422 (1999). Coordinated interactions or “cross-talk” betweenthese two protein kinases in regulation of mucin secretion has onlyrecently been demonstrated to involve the MARCKS proteins. See, Li etal., Journal of Biological Chemistry, 276: 40982-40990 (2001). However,signaling events downstream of the coordinated action of these proteinkinases that ultimately leads to the exocytotic release of mucingranules have not been fully elucidated.

MARCKS, a protein of approximately 82 kD, has threeevolutionarily-conserved regions (Aderem et al., Nature 1988;332:362-364; Thelen et al., Nature 1991; 351:320-322; Hartwig et al.,Nature 1992; 356:618-622; Seykora et al., J Biol Chem 1996;271:18797-18802): an N-terminus, a phosphorylation site domain (PSD),and a multiple homology 2 (MH2) domain. The N-terminus, a 24 amino acidsequence with a myristic acid moiety attached to a terminal glycineresidue is involved in binding of MARCKS to membranes (Seykora et al., JBiol Chem 1996; 271:18797-18802) and possibly to calmodulin (Matsubaraet al., J Biol Chem 2003; 278:48898-48902). This 24 amino acid sequenceis known as the MANS peptide. The MANS peptide and active fragmentsthereof, can compete with native MARCKS in cells for membrane binding.Involvement of MARCKS protein in release of inflammatory mediators fromthe granules of infiltrating leukocytes is relevant to inflammation indiseases in all tissues and organs, including lung diseasescharacterized by airway inflammation, such as asthma, COPD and cysticfibrosis. However, inflammation and mucus secretion in the airways aretwo separate and independent processes (Li et al., J Biol Chem 2001;276:40982-40990; Singer et al., Nat Med 2004; 10:193-196). While mucusproduction and secretion can be provoked by a number of factors,including mediators released by inflammatory cells, there is no knowndirect link between excess mucus and inflammation.

SUMMARY OF THE INVENTION

The invention relates to a new use for the 24 amino acid, myristoylatedpolypeptide, also known as the MANS peptide. The invention also relatesto a new method for blocking any cellular secretory process, especiallythose that involve the release of inflammatory mediators frominflammatory cells, whose stimulatory pathways involve the proteinkinase C (PKC) substrate MARCKS protein and release of contents fromintracellular vesicles or granules.

The present invention is directed to a method of inhibiting theexocytotic release of at least one inflammatory mediator from at leastone inflammatory cell comprising contacting the at least oneinflammatory cell, which cell comprises at least one inflammatorymediator contained within a vesicle inside the cell, with at least onepeptide selected from the group consisting of a MANS peptide and anactive fragment thereof in an effective amount to reduce the release ofthe inflammatory mediator from the inflammatory cell as compared to therelease of the inflammatory mediator from the same type of inflammatorycell that would occur in the absence of the at least one peptide.

The present invention is further directed to a method of inhibiting therelease of at least one inflammatory mediator from at least oneinflammatory cell in a tissue or fluid of a subject comprising theadministration to the subject's tissue and/or fluid, which comprises atleast one inflammatory cell comprising at least one inflammatorymediator contained within a vesicle inside the cell, a therapeuticallyeffective amount of a pharmaceutical composition comprising at least onepeptide selected from the group consisting of a MANS peptide and anactive fragment thereof in a therapeutically effective amount to reducethe release of the inflammatory mediator from at least one inflammatorycell as compared to release of the inflammatory mediator from at leastone of the same type of inflammatory cell that would occur in theabsence of the at least one peptide. More specifically, inhibiting therelease of an inflammatory mediator comprises blocking or reducing therelease of an inflammatory mediator from the inflammatory cell.

More particularly, the present invention includes a method of reducinginflammation in a subject comprising the administration of atherapeutically effective amount of a pharmaceutical compositioncomprising a MANS peptide (i.e., N-myristoyl-GAQFSKTAAKGEAAAERPGEAAV(SEQ ID NO: 1)) or an active fragment thereof. The active fragment is atleast six amino acids in length. As used herein, an “active fragment” ofa MARCKS protein is one that affects (inhibits or enhances) the MARCKSprotein-mediated release. An active fragment can be selected from thegroup consisting of N-myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO: 3);N-myristoyl-GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 4);N-myristoyl-GAQFSKTAAKGEAAAERPGE (SEQ ID NO: 5);N-myristoyl-GAQFSKTAAKGEAAAERPG (SEQ ID NO: 6);N-myristoyl-GAQFSKTAAKGEAAAERP (SEQ ID NO: 7);N-myristoyl-GAQFSKTAAKGEAAAER (SEQ ID NO: 8);N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID NO: 9); N-myristoyl-GAQFSKTAAKGEAAA(SEQ ID NO: 10); N-myristoyl-GAQFSKTAAKGEAA (SEQ ID NO: 11);N-myristoyl-GAQFSKTAAKGEA (SEQ ID NO: 12); N-myristoyl-GAQFSKTAAKGE (SEQID NO: 13); N-myristoyl-GAQFSKTAAKG (SEQ ID NO: 14);N-myristoyl-GAQFSKTAAK (SEQ ID NO: 15); N-myristoyl-GAQFSKTAA (SEQ IDNO: 16); N-myristoyl-GAQFSKTA (SEQ ID NO: 17); N-myristoyl-GAQFSKT (SEQID NO: 18); and N-myristoyl-GAQFSK (SEQ ID NO: 19). The presence of thehydrophobic N-terminal myristate moiety in these peptides can enhancetheir compatibility with and presumably their permeability to plasmamembranes, and potentially enable the peptides to be taken up by cells.The hydrophobic insertion of myristate into a bilayer can provide apartition coefficient or apparent association constant with lipids of upto 10⁴ M⁻¹ or a unitary Gibbs free binding energy of about 8 kcal/mol(see, for example, Peitzsch, R. M., and McLaughlin, S. 1993, Binding ofacylated peptides and fatty acids to phospholipid vesicles: pertinenceto myristoylated proteins. Biochemistry. 32: 10436-10443) which issufficient, at least in part, to permit a partitioning of the MANSpeptide and of myristoylated MANS peptide fragments as described hereininto the plasma membrane of a cell while additional functional groupsand their interactions within the MANS peptide (which is myristoylated)and within myristoylated MANS peptide fragments can potentiate theirrelative membrane permeabilities. The fragments can each exhibitpartition coefficients and membrane affinities that are representativeof their respective structure. The fragments can be prepared by methodsof peptide synthesis known in the art, such as by solid phase peptidesynthesis (see, for example, the methods described in Chan, Weng C. andWhite, Peter D. Eds., Fmoc Solid Phase Peptide Synthesis: A PracticalApproach, Oxford University Press, New York, N.Y. (2000); andLloyd-Williams, P. et al. Chemical Approaches to the Synthesis ofPeptides and Proteins (1997)) and purified by methods known in the art,such as by high pressure liquid chromatography. Molecular weight of eachpeptide can be confirmed by mass spectroscopy with each showing a peakwith an appropriate molecular mass. Efficacy of the individual peptidesand of combinations of individual peptides (for example, combinations of2 of the peptides, combinations of 3 of the peptides, combinations of 4of the peptides) in the methods of this disclosure can be readilydetermined without undue experimentation using the procedures describedin the examples disclosed herein. A preferred combination will comprisetwo of the peptides; a preferred molar ratio of the peptides can be from50:50 to 99.99 to 0.01, which ratio can be readily determined using theprocedures described in the examples disclosed herein.

Preferably the MANS peptide or active fragment thereof is contained in apharmaceutical composition which is useful to block inflammation. Thepresent invention also includes methods for regulating a cellularsecretory process in a subject comprising the administration of atherapeutically effective amount of a compound comprising a MANS peptideor an active fragment thereof, that regulates an inflammatory mediatorin a subject. The administration is generally selected from the groupconsisting of topical administration, parenteral administration, rectaladministration, pulmonary administration, inhalation and nasal or oraladministration, wherein pulmonary administration generally includeseither an aerosol, a dry powder inhaler, a metered dose inhaler, or anebulizer.

Administration of a composition comprising a degranulation-inhibitingamount of the MANS peptide or a degranulation-inhibiting amount of anactive fragment thereof, such as a pharmaceutical composition of theMANS peptide or an active fragment thereof, for human or animal useprovides the MANS peptide or active fragment thereof at least to thesite in or on a tissue or to a fluid-containing or mucus-containinglayer in contact with the surface of a tissue where an inflammatorygranulocytic cell resides or into which an inflammatory granulocyticcell will invade, thus enabling the MANS peptide or an active fragmentthereof to contact the inflammatory granulocytic cell. In one aspect,administration of such a composition can be made at the first onset orfirst detection of inflammation or first perception of inflammation bythe human or animal or at the first perceptible change in the level ofinflammation in a human or animal to reduce the amount of inflammationthat would otherwise occur in the absence of the MANS peptide or activefragment thereof. In another aspect, administration can be made duringan ongoing inflammation of a tissue in the human or animal to reduce theamount of additional inflammation that would otherwise occur in theabsence of the MANS peptide or active fragment thereof. While the amountand frequency of dose can be determined by clinical evaluation and be afunction of the disease or source of inflammation and the extent oftissue involved and the age and size of the patient, it is anticipatedthat dosing of a pharmaceutical composition can be repeated after 3 to 8hours, preferably after 6 to 8 hours after the first administration ofthe pharmaceutical composition.

The present invention also includes methods of reducing inflammation ina subject comprising the administration of a therapeutically effectiveamount of a compound that inhibits the MARCKS-related release ofinflammatory mediators, whereby the release of at least one inflammatorymediator in the subject is reduced compared to that which would occur inthe absence of said treatment. As used herein “reducing” generally meansa lessening of the effects of inflammation. Preferably, inflammatorymediators are inhibited or blocked by the methods disclosed.

Another embodiment of the present invention includes methods of reducinginflammation in a subject comprising administering a therapeuticallyeffective amount of a compound that inhibits the MARCKS-related releaseof inflammatory mediators, whereby the inflammation in the subject isreduced compared to that which would occur in the absence of saidtreatment. The present invention also discloses methods of reducing orinhibiting inflammation in a subject comprising the administration of atherapeutically effective amount of a MANS peptide or an active fragmentthereof effective to modulate an inflammatory mediator at theinflammation site. The term “inhibiting” means a reduction in the amountof inflammatory mediator secretion. The term “completely inhibiting”means a reduction to zero in the amount of inflammatory mediatorsecretion. Again, as stated above, the active fragment is at least sixamino acids in length. The term “exocytotic process” means exocytosis,i.e., a process of cellular secretion or excretion in which substancescontained in a vesicle, which vesicle resides inside a cell, aredischarged from the cell by fusion of the vesicular membrane of thevesicle with the outer cell membrane. “Degranulation” means the releaseof cellular granule contents. The term “degranulation-inhibiting” meansa reduction in the release of the inflammatory mediators containedwithin the granules of the inflammatory cell. Thus, adegranulation-inhibiting amount of the MANS peptide and/or an activefragment thereof is the amount of these peptides that is sufficient toreduce the release of the inflammatory mediators contained in thegranules as compared to release in the absence of the same peptide.

MANS peptide and active fragments thereof can be useful in theprevention or reduction in amount of inflammation in a tissue in ananimal caused by inflammatory mediators. MANS peptide and activefragments thereof can be useful in the prevention or reduction in amountof tissue damage in an animal produced or caused by inflammatorymediators.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are bar graphs illustrating mucin hypersecretion by NHBEcells is maximized by activation of both PKC and PKG.

FIGS. 2A-2B demonstrate that the MARCKS protein is a key component ofthe mucin secretory pathway.

FIGS. 3A-3C depicts a gel illustrating that an antisense oligonucleotidedirected against MARCKS down-regulates MARCKS expression and attenuatesmucin hypersecretion.

FIGS. 4A-4B illustrate that PKC-dependent phosphorylation releasesMARCKS from the plasma membrane to the cytoplasm.

FIGS. 5A-5C show that PKG induces dephosphorylation of MARCKS byactivating PP2A.

FIG. 6 depicts bar graphs that demonstrate that PP2A is an essentialcomponent of the mucin secretory pathway.

FIG. 7 is a gel that illustrates that MARCKS associates with actin andmyosin in the cytoplasm.

FIG. 8 depicts a signaling mechanism controlling mucin secretion byhuman airway epithelial cells.

FIG. 9 is a bar graph depicting the ability of MANS peptide to blocksecretion of myloperoxidase from isolated canine neutrophils.

FIG. 10 is a bar graph depicting the ability of MANS peptide to blocksecretion of myloperoxidase from isolated human neutrophils.

FIG. 11 is a bar graph showing that PMA stimulates a small increase inMPO secretion from LPS-stimulated human neutrophils which is enhanced ina concentration-dependent manner by co-stimulation with 8-Br-cGMP.

FIG. 12 is a bar graph showing that 8-Br-cGMP simulation has littleeffect on MPO secretion from LPS-stimulated human neutrophils until aco-stimulation with PMA occurs in a concentration-dependent manner.

FIG. 13 is a bar graph showing that PMA stimulates a small increase inMPO secretion from LPS-stimulated canine neutrophils which is enhancedin a concentration-dependent manner by co-stimulation with 8-Br-cGMP.

FIG. 14 is a bar graph showing that 8-Br-cGMP simulation has littleeffect on MPO secretion from LPS-stimulated canine neutrophils until aco-stimulation with PMA occurs in a concentration-dependent manner.

FIG. 15 is a bar graph showing that co-stimulation with PMA+8-Br-cGMP isrequired for maximal MPO secretion from LPS-stimulated canineneutrophils.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which preferred embodiments ofthe invention are illustrated. This invention may, however, be embodiedin different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. The use of the words “a” or“an” herein to describe any aspect of the present invention is to beinterpreted as indicating one or more.

The present invention is directed to a method of inhibiting theexocytotic release of at least one inflammatory mediator from at leastone inflammatory cell comprising contacting the at least oneinflammatory cell, which cell comprises at least one inflammatorymediator contained within a vesicle inside the cell, with at least onepeptide selected from the group consisting of a MANS peptide and anactive fragment thereof in an effective amount to reduce the release ofthe inflammatory mediator from the inflammatory cell as compared to therelease of the inflammatory mediator from the same type of inflammatorycell that would occur in the absence of the at least one peptide.

The present invention is further directed to a method of inhibiting therelease of at least one inflammatory mediator from at least oneinflammatory cell in a tissue or fluid of a subject comprising theadministration to the subject's tissue and/or fluid, which comprises atleast one inflammatory cell comprising at least one inflammatorymediator contained within a vesicle inside the cell, a therapeuticallyeffective amount of a pharmaceutical composition comprising at least onepeptide selected from the group consisting of a MANS peptide and anactive fragment thereof in a therapeutically effective amount to reducethe release of the inflammatory mediator from at least one inflammatorycell as compared to release of the inflammatory mediator from at leastone of the same type of inflammatory cell that would occur in theabsence of the at least one peptide. More specifically, reducing therelease of an inflammatory mediator comprises blocking or inhibiting themechanism that releases an inflammatory mediator from the inflammatorycell.

The MANS peptide used in the present methods described above comprisesSEQ ID NO:1. The active fragment useful in the present inventioncomprises at least one myristoylated N-terminal fragment of SEQ ID NO:1which comprises at least six amino acids, wherein the first amino acidof said fragment begins at the N-terminal glycine of SEQ ID NO:1. Morespecifically, the active fragment can be selected from the groupconsisting of N-myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO: 3);N-myristoyl-GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 4);N-myristoyl-GAQFSKTAAKGEAAAERPGE (SEQ ID NO: 5);N-myristoyl-GAQFSKTAAKGEAAAERPG (SEQ ID NO: 6);N-myristoyl-GAQFSKTAAKGEAAAERP (SEQ ID NO: 7);N-myristoyl-GAQFSKTAAKGEAAAER (SEQ ID NO: 8);N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID NO: 9); N-myristoyl-GAQFSKTAAKGEAAA(SEQ ID NO: 10); N-myristoyl-GAQFSKTAAKGEAA (SEQ ID NO: 11);N-myristoyl-GAQFSKTAAKGEA (SEQ ID NO: 12); N-myristoyl-GAQFSKTAAKGE (SEQID NO: 13); N-myristoyl-GAQFSKTAAKG (SEQ ID NO: 14);N-myristoyl-GAQFSKTAAK (SEQ ID NO: 15); N-myristoyl-GAQFSKTAA (SEQ IDNO: 16); N-myristoyl-GAQFSKTA (SEQ ID NO: 17); N-myristoyl-GAQFSKT (SEQID NO: 18); and N-myristoyl-GAQFSK (SEQ ID NO: 19).

The present invention is directed to the contact and/or administrationof the peptide described above and throughout the specification with anyknown inflammatory cell that may be contained in the tissue or fluid ofa subject which contains at least one inflammatory mediator containedwithin a vesicle inside the cell. The inflammatory cell is preferably aleukocyte, more preferably a granulocyte, which can be furtherclassified as a neutrophil, a basophil, an eosinophil or a combinationthereof. The inflammatory cells contacted in the present method may alsobe a monocyte/macrophage.

The present invention is directed to reducing the release ofinflammatory mediators contained within the vesicles of inflammatorycells and these inflammatory mediators are selected from the groupconsisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO), majorbasic protein (MBP), lysozyme, granzyme, histamine, proteoglycan,protease, a chemotactic factor, cytokine, a metabolite of arachidonicacid, defensin, bactericidal permeability-increasing protein (BPI),elastase, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase,alpha-mannosidase, phospholipase A₂, chondroitin-4-sulphate, proteinase3, lactoferrin, collagenase, complement activator, complement receptor,N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, lamininreceptor, cytochrome b₅₅₈, monocyte-chemotactic factor, histaminase,vitamin B12 binding protein, gelatinase, plasminogen activator,beta-D-glucuronidase, and a combination thereof. Preferably, theseinflammatory mediators are selected from the group consisting ofmyeloperoxidase (MPO), eosinophil peroxidase (EPO), major basic protein(MBP), lysozyme, granzyme and a combination thereof.

The present invention contacts an effective amount of the peptide withan inflammatory cell, wherein the effective amount is defined as adegranulation-inhibiting amount of MANS peptide or an active fragmentthereof that reduces the amount of an inflammatory mediator releasedfrom at least one inflammatory cell from about 1% to about 99% ascompared to the amount released from at least one inflammatory cell inthe absence of MANS peptide or an active fragment thereof. Morepreferably, this effective amount of the contacted peptide comprises adegranulation-inhibiting amount of MANS peptide or an active fragmentthereof that reduces the amount of an inflammatory mediator releasedfrom at least one inflammatory cell from between about 5-50% to about99% as compared to the amount released from at least one inflammatorycell in the absence of MANS peptide or an active fragment thereof.

The present invention in one embodiment is directed to theadministration of at least one peptide comprising a MANS peptide and anactive fragment thereof in a therapeutically effective amount intotissue or fluid of a subject where the subject is afflicted by arespiratory disease, which is preferably asthma, chronic bronchitis orCOPD. In a further embodiment, the subject may be afflicted by a boweldisease, a skin disease, an autoimmune disease, a pain syndrome, andcombinations thereof. The bowel disease may be ulcerative colitis,Crohn's disease or irritable bowel syndrome. The subject may beafflicted with a skin disease, such as rosacea, eczema, psoriasis orsevere acne. The subject may also be afflicted with arthritis, such asrheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus.Subjects afflicted by cystic fibrosis may also be treated by the presentmethod and peptides. The present method is preferably useful for thetreatment of subjects, such as mammals, and preferably humans, canines,equines and felines.

The present method of treatment of subjects is by the administration ofone or more peptides including the MANS peptide or an active fragmentdescribed herein to include topical administration, parenteraladministration, rectal administration, pulmonary administration, nasaladministration, or oral administration. More specifically, pulmonaryadministration is selected from the group of aerosol, dry powderinhaler, metered dose inhaler, and nebulizer. Additionally, thedisclosed method may further comprise the administration to the subjectof a second molecule selected from the group consisting of anantibiotic, an antiviral compound, an antiparasitic compound, ananti-inflammatory compound, and an immunosuppressant.

In one aspect, the invention relates to a method of administering apharmaceutical composition. The pharmaceutical composition comprises atherapeutically effective amount of a known compound and apharmaceutically acceptable carrier. A “therapeutically effective”amount as used herein is an amount of a compound that is sufficient toameliorate symptoms exhibited by a subject. The therapeuticallyeffective amount will vary with the age and physical condition of thepatient, the severity of the condition of the patient being treated, theduration of the treatment, the nature of any concurrent treatment, thepharmaceutically acceptable carrier used and like factors within theknowledge and expertise of those skilled in the art. Pharmaceuticallyacceptable carriers are preferably solid dosage forms such as tablets orcapsules. Liquid preparations for oral administration also may be usedand may be prepared in the form of syrups or suspensions, e.g.,solutions containing an active ingredient, sugar, and a mixture ofethanol, water, glycerol, and propylene glycol. If desired, such liquidpreparations may include one or more of following: coloring agents,flavoring agents, and saccharin. Additionally, thickening agents such ascarboxymethylcellulose also may be used as well as other acceptablecarriers, the selection of which are known in the art.

As stated above, the present invention relates to methods for regulatingcellular secretory processes, especially those releasing inflammatorymediators from inflammatory cells. As used herein, the term “regulating”means blocking, inhibiting, decreasing, reducing, increasing, enhancingor stimulating. A number of cellular secretory processes involve therelease of contents from membrane-bound vesicles or granules withincells A membrane-bound vesicle or granule is defined as an intracellularparticle, which is primarily vesicular (or a vesicle inside a cell) andwhich contains stored material that can be secreted. Some of thecontents of these vesicles, such as those contained in inflammatorycells, have been found to be responsible for a variety of pathologies innumerous mammalian tissues. Some of the effects of these secretionsappear to include damage of previously healthy tissue duringinflammation. This invention provides a means of blocking secretion fromany membrane-bound vesicle, including those found in inflammatory cells,by targeting a specific molecule important in the intracellularsecretory pathway with a synthetic peptide. This approach may be oftherapeutic importance for the treatment of a wide variety ofhypersecretory and inflammatory conditions in humans and animals.

More specifically, the present invention targets inflammatory cells thatcontain the inflammatory mediators in one or more granules or vesicleswithin the cells' cytoplasm. The cells are contacted with one or morepeptides that are selected from the MANS peptide or an active fragmentthereof, all of which are described in detail herein. Preferably thecontact of the peptide with the inflammatory cell is via administrationto a subject afflicted by or suffering from a disease in which theseinflammatory cells are present in specific tissue or fluid within thetissue. Upon administration or contact of the peptide with the cell, thepeptide competitively competes for and competitively inhibits thebinding of the native MARCKS protein to the membrane of theintracellular granules or vesicles which contain the inflammatorymediators. As a result of blocking the binding of the MARCKS protein tothe vesicles in the inflammatory cells, these vesicles in these cells donot move to the plasma membrane of the cells as they would normally dowhen stimulated to exocytotically release their contents of inflammatorymediators out of the cells. Thus, the method of the present inventioninhibits the movement of the vesicles to the cells' plasma membrane,which in turn, reduces the release of the inflammatory mediators fromthe inflammatory cells. The amount of inflammatory mediators releasedfrom the cells over time is reduced because both the rate of release andthe amount of release of the mediators from the inflammatory cells isdependent upon the concentration of the peptide administered andcontacted with the inflammatory cells.

One benefit of the present invention is that it may combine a therapythat includes the direct blocking of mucus secretion with a uniqueanti-inflammatory therapy. A benefit of the present invention overcurrent anti-inflammation therapies that affect a general suppression ofthe immune system is that the peptide is thought to block secretion ofonly intracellular components secreted from inflammatory cells. Thus,many aspects of the immune system should still function even with theinhibition of the inflammatory mediators.

The compounds of the invention may regulate, i.e. block, inflammatorymediator release from cells. This inhibition of release of inflammatorymediators is an attractive means for preventing and treating a varietyof disorders, e.g., diseases and pathological conditions involvinginflammation. Thus, the compounds of the invention may be useful for thetreatment of such conditions. These encompass airway diseases andchronic inflammatory diseases including, but not limited to,osteoarthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn'sdisease, ulcerative colitis, psoriasis, graft versus host disease,systemic lupus erythematosus and insulin-dependent diabetes mellitus.The compounds of the invention can also be used to treat other disordersassociated with the activity of elevated levels of proinflammatorymediators and enzymes such as responses to various infectious agents anda number of diseases of autoimmunity such as rheumatoid arthritis, toxicshock syndrome, diabetes and inflammatory bowel diseases.

Uses of the peptide and methods of the invention include therapies tocombat inflammation along with therapies that will combine theanti-inflammatory activity of the peptide with its ability to blockmucus secretion. Diseases that may be treated by the peptide's abilityto block both inflammation and mucus secretion include but are notlimited to inflammatory bowel diseases, digestive disorders (i.e.,inflamed gall bladder, Menetier's disease) and inflammatory airwaydiseases. The peptide may also be used to block release of excessinsulin from pancreatic islet cells.

Other proinflammatory mediators have been correlated with a variety ofdisease states that correlate with influx of neutrophils into sites ofinflammation or injury. Blocking antibodies have been demonstrated asuseful therapies against the neutrophil-associated tissue injury inacute inflammation (Harada et al., 1996, Molecular Medicine Today 2,482). Cells other than neutrophils that may release inflammatorymediators include other leukocytes, such as basophils, eosinophils,monocytes and lymphocytes, and therapies may be directed againstsecretion from these cells. Neutrophils, eosinophils, and basophils areeach a type of granulocyte, i.e., a leukocyte that has granules in itscytoplasm. Leukocytes synthesize a number of inflammatory mediators thatare packaged and stored in cytoplasmic granules. Among these mediatorsare, for example, myeloperoxidase [MPO] in neutrophils (Borregaard N,Cowland J B. Granules of the human neutrophilic polymorphonuclearleukocyte. Blood 1997; 89:3503-3521), eosinophil peroxidase [EPO] andmajor basic protein [MBP] in eosinophils (Gleich G J. Mechanisms ofeosinophil-associated inflammation. J Allergy Clin Immunol 2000;105:651-663), lysozyme in monocytes/macrophages (Hoff T, Spencker T,Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on theTPA-induced monocytic differentiation. J Leukoc Biol 1992; 52:173-182;Balboa M A, Saez Y, Balsinde J. Calcium-independent phospholipase A2 isrequired for lysozyme secretion in U937 promonocytes. J Immunol 2003;170:5276-5280), and granzyme in natural killer (NK) cells and cytotoxiclymphocytes (Bochan M R, Goebel W S, Brahmi Z. Stably transfectedantisense granzyme B and perforin constructs inhibit humangranule-mediated lytic ability. Cell Immunol 1995; 164:234-239; Gong JH., Maki G, Klingemann H G. Characterization of a human cell line(NK-92) with phenotypical and functional characteristics of activatednatural killer cells. Leukemia 1994; 8:652-658; Maki G, Klingemann H G,Martinson J A, Tam Y K. Factors regulating the cytotoxic activity of thehuman natural killer cell line, NK-92. J Hematother Stem Cell Res 2001;10:369-383; and Takayama H, Trenn G, Sitkovsky M V. A novel cytotoxic Tlymphocyte activation assay. J Immunol Methods 1987; 104:183-1907-10).These mediators can be released at sites of injury and can contribute toinflammation and repair, such as in the lung and elsewhere, as a resultof the infiltration of these cells to the tissue site of injury ordisease. Leukocytes release these granules via an exocytotic mechanism(Burgoyne R D, Morgan A. Secretory granule exocytosis. Physiol Rev 2003;83:581-632; Logan M R, Odemuyiwa S O, Moqbel R. Understanding exocytosisin immune and inflammatory cells: the molecular basis of mediatorsecretion. J Allergy Clin Immunol 2003; 111: 923-932),

Mast cells, which usually do not circulate in the blood stream, andbasophils contain secretory cytoplasmic granules which store and canrelease, upon cell activation, preformed inflammatory (anaphylactic)mediators, such as histamine; proteoglycans, such as heparin andchondroitin sulphate; proteases such as tyrptase, chymase,carboxypeptidase, and cathepsin G-like protease; chemotactic factors,cytokines and metabolites of arachidonic acid that act on thevasculature, smooth muscle, connective tissue, mucous glands andinflammatory cells.

Neutrophils, also known as polymorphonuclear leukocytes (PMN), comprise50 to 60% of the total circulating leukocytes. Neutrophils act againstinfectious agents, such as bacteria, fungi, protozoa, viruses, virallyinfected cells, as well as tumor cells, that penetrate the body'sphysical barriers at sites of infection or injury. Neutrophils maturethrough six morphological stages: myeloblast, promyeloblast, myelocyte,metamyelocyte, non-segmented (band) neutrophil, and segmented(functionally active) neutrophil.

In neutrophils, inflammatory mediators are stored in primary(azurophil), secondary (specific), and tertiary (gelatinase) granules,as well as in secretory vesicles. Among numerous mediators ofinflammation, primary (azurophil) granules contain myeloperoxidase(MPO), lysozyme, defensins, bactericidal permeability-increasing protein(BPI), elastase, cathepsin G, cathepsin B, cathepsin D,beta-D-glucuronidase, alpha-mannosidase, phospholipase A₂,chondroitin-4-sulphate, and proteinase 3 (see, for example, Hartwig J H,Thelen M, Rosen A, Janmey P A, Nairn A C, Aderem A. MARCKS is an actinfilament crosslinking protein regulated by protein kinase C andcalcium-calmodulin. Nature 1992; 356:618-622); secondary (specific)granules contain lysozyme, lactoferrin, collagenase, complementactivator, phospholipase A₂, complement receptors, e.g., CR3, CR4,N-formylmethionyl-leucyl-phenylalanine (FMLP) receptors, lamininreceptors, cytochrome b₅₅₈, monocyte-chemotactic factor, histaminase,and vitamin B12 binding protein; and small storage granules containgelatinase, plasminogen activator, cathepsin B, cathepsin D,beta-D-glucuronidase, alpha-mannosidase, and cytochrome b₅₅₈.

Neutrophil granules contain antimicrobial or cytotoxic substances,neutral proteinases, acid hydrolases and a pool of cytoplasmic membranereceptors. Among azurophil granule constituents myeloperoxidase (MPO) isa critical enzyme in the conversion of hydrogen peroxide to hypochlorousacid. Together with hydrogen peroxide and a halide cofactor it forms aneffective microbicidal and cytotoxic mechanism of leukocytes—themyeloperoxidase system.

Defensins, which constitute 30 to 50% of azurophilic granule protein,are small (molecule weight<4000) potent antimicrobial peptides that arecytotoxic to a broad range of bacteria, fungi and some viruses. Theirtoxicity may be due to membrane permeabilization of the target cellwhich is similar to other channel-forming proteins (perforins).

Bacterial permeability-increasing (BPI) protein is a member ofperforins. It is highly toxic to gram-negative bacteria but not togram-positive bacteria or fungi and can also neutralize endotoxin, thetoxic lipopolysaccharide component of gram-negative bacterial cellenvelope.

Lactoferrin sequesters free iron, thereby preventing the growth ofingested microorganisms that survive the killing process and increasesbacterial permeability to lysozyme.

Serine proteases such as elastase and cathepsin G hydrolyze proteins inbacterial cell envelopes. Substrates of granulocyte elastase includecollagen cross-linkages and proteoglycans, as well as elastin componentsof blood vessels, ligaments, and cartilage. Cathepsin D cleavescartilage proteoglycans, whereas granulocyte collagenases are active incleaving type I and, to a lesser degree, type III collagen from bone,cartilage, and tendon. Collagen breakdown products have chemotacticactivity for neutrophils, monocytes, and fibroblasts.

Regulation of tissue destructive potential of lysosomal proteases ismediated by protease inhibitors such as alpha2-macroglobulin andalpha1-antiprotease. These antiproteases are present in serum andsynovial fluids. They may function by binding to and covering the activesites of proteases. Protease-antiprotease imbalance can be important inthe pathogenesis of emphysema.

Azurophil granules function predominantly in the intracellular milieu(in the phagolysosomal vacuole), where they are involved in the killingand degradation of microorganisms. Neutrophil specific granules aresusceptible to release their contents extracellularly and have animportant role in initiating inflammation. Specific granules representan intracellular reservoir of various plasma membrane componentsincluding cytochrome b (component of NADPH oxidase, an enzymeresponsible for the production of superoxide), receptors for complementfragment iC3b (CR3, CR4), for laminin, and formylmethionyl-peptidechemoattractants. In addition to others, there is histaminase which isrelevant for the degradation of histamine, vitamin binding protein, andplasminogen activator which is responsible for plasmin formation andcleavage of C5a from C5.

The importance of neutrophil granules in inflammation is apparent fromstudies of several patients with congenital abnormalities of thegranules. Patients with Chédiak-Higashi syndrome have a profoundabnormality in the rate of establishment of an inflammatory response andhave abnormally large lysosomal granules. The congenital syndrome ofspecific granule deficiency is an exceedingly rare disordercharacterized by diminished inflammatory responses and severe bacterialinfections of skin and deep tissues.

Although mechanisms regulating exocytotic secretion of these granulesare only partially understood, several key molecules in the process havebeen identified, including intracellular Ca2+ transients (Richter et al.Proc Natl Acad Sci USA 1990; 87:9472-9476; Blackwood et al., Biochem J1990; 266:195-200), G proteins, tyrosine and protein kinases (PK,especially PKC) (Smolen et al., Biochim Biophys Acta 1990; 1052:133-142;Niessen et al., Biochim. Biophys. Acta 1994; 1223:267-273; Naucler etal., Pettersen et al., Chest 2002; 121; 142-150), Rac2 (Abdel-Latif etal., Blood 2004; 104:832-839; Lacy et al., J Immunol 2003;170:2670-2679) and various SNARE's, SNAP's and VAMP's (Sollner et al.,Nature 1993; 362: 318-324; Lacy, Pharmacol Ther 2005; 107:358-376).

SNARE (Soluble N-ethylmaleimide attachment protein receptor) proteinsare a family of membrane-associated proteins characterized by analpha-helical coiled-coil domain called the SNARE motif (Li et al.,Cell. Mol. Life. Sci. 60: 942-960 (2003)). These proteins are classifiedas v-SNAREs and t-SNAREs based on their localization on vesicle ortarget membrane; another classification scheme defines R-SNAREs andQ-SNAREs, as based on the conserved arginine or glutamine residue in thecentre of the SNARE motif. SNAREs are localized to distinct membranecompartments of the secretory and endocytic trafficking pathways, andcontribute to the specificity of intracellular membrane fusionprocesses. The t-SNARE domain consists of a 4-helical bundle with acoiled-coil twist. The SNARE motif contributes to the fusion of twomembranes. SNARE motifs fall into four classes: homologues of syntaxin1a (t-SNARE), VAMP-2 (v-SNARE), and the N- and C-terminal SNARE motifsof SNAP-25. One member from each class may interact to form a SNAREcomplex. The SNARE motif is found in the N-terminal domains of certainsyntaxin family members such as syntaxin 1a, which is required forneurotransmitter release (Lerman et al., Biochemistry 39: 8470-8479(2000)), and syntaxin 6, which is found in endosomal transport vesicles(Misura et al., Proc. Natl. Acad. Sci. U.S.A. 99: 9184-9189 (2002)).

SNAP-25 (synaptosome-associated protein 25 kDa) proteins are componentsof SNARE complexes, which may account for the specificity of membranefusion and to directly execute fusion by forming a tight complex (theSNARE or core complex) that brings the synaptic vesicle and plasmamembranes together. The SNAREs constitute a large family of proteinsthat are characterized by 60-residue sequences known as SNARE motifs,which have a high propensity to form coiled coils and often precedecarboxy-terminal transmembrane regions. The synaptic core complex isformed by four SNARE motifs (two from SNAP-25 and one each fromsynaptobrevin and syntaxin 1) that are unstructured in isolation butform a parallel four-helix bundle on assembly. The crystal structure ofthe core complex has revealed that the helix bundle is highly twistedand contains several salt bridges on the surface, as well as layers ofinterior hydrophobic residues. A polar layer in the centre of thecomplex is formed by three glutamines (two from SNAP-25 and one fromsyntaxin 1) and one arginine (from synaptobrevin) (Rizo et al., Nat RevNeurosci 3: 641-653 (2002)). Members of the SNAP-25 family contain acluster of cysteine residues that can be palmitoylated for membraneattachment (Risinger et al., J. Biol. Chem. 268: 24408-24414 (1993)).

The major role of neutrophils is to phagocytose and destroy infectiousagents. They also limit the growth of some microbes, prior to onset ofadaptive (specific) immunological responses. Although neutrophils areessential to host defense, they have also been implicated in thepathology of many chronic inflammatory conditions and inischemia-reperfusion injury. Hydrolytic enzymes of neutrophil origin andoxidatively inactivated protease inhibitors can be detected in fluidisolated from inflammatory sites. Under normal conditions, neutrophilscan migrate to sites of infection without damage to host tissues.However, undesirable damage to a host tissue can sometimes occur. Thisdamage may occur through several independent mechanisms. These includepremature activation during migration, extracellular release of toxicproducts during the killing of some microbes, removal of infected ordamage host cells and debris as a first step in tissue remodeling, orfailure to terminate acute inflammatory responses. Ischemia-reperfusioninjury is associated with an influx of neutrophils into the affectedtissue and subsequent activation. This may be triggered by substancesreleased from damaged host cells or as a consequence of superoxidegeneration through xantine oxidase.

Under normal conditions, blood may contain a mixture of normal, primed,activated and spent neutrophils. In an inflammatory site, mainlyactivated and spent neutrophils are present. Activated neutrophils haveenhanced production of reactive oxygen intermediates (ROI). Asubpopulation of neutrophils with the enhanced respiratory burst hasbeen detected in the blood of people with an acute bacterial infectionand patients with the adult respiratory distress syndrome (ARDS). Thisis an example of a neutrophil paradox. Neutrophils have been implicatedin the pathology of this condition because of the large influx of thesecells into the lung and the associated tissue damage caused by oxidantsand hydrolytic enzymes released from activated neutrophils. Theimpairment of neutrophil microbicidal activity that occurs as the ARDSworsens may be a protective response on the part of the host, which isinduced locally by inflammatory products.

The acute phase of thermal injury is also associated with neutrophilactivation, and this is followed by a general impairment in variousneutrophil functions. Activation of neutrophils by immune complexes insynovial fluid contributes to the pathology of rheumatoid arthritis.Chronic activation of neutrophils may also initiate tumor developmentbecause some ROI generated by neutrophils damage DNA and proteasespromote tumor cell migration. In patients suffering from severe burns, acorrelation has been established between the onset of bacterialinfection and reduction in the proportion and absolute numbers ofneutrophils positive for antibody and complement receptors. Oxidants ofneutrophil origin have also been shown to oxidize low-densitylipoproteins (LDL), which are then more effectively bound to the plasmamembrane of macrophages through specific scavenger receptors. Uptake ofthese oxidized LDL by macrophages may initiate atherosclerosis. Inaddition, primed neutrophils have been found in people with essentialhypertension, Hodgkin's disease, inflammatory bowel disease, psoriasis,sarcoidosis, and septicemia, where priming correlates with highconcentrations of circulating TNF-alpha (cachectin).

Hydrolytic damage to host tissue and therefore chronic inflammatoryconditions may occur when antioxidant and antiprotease screens areoverwhelmed. Antiprotease deficiency is thought to be responsible forthe pathology of emphysema. Many antiproteases are members of the serineprotease inhibitor (SERPIN) family. Although the circulation is rich inantiproteases, these large proteins may be selectively excluded at sitesof inflammation because neutrophils adhere to their targets. Oxidativestress may initiate tissue damage by reducing the concentration ofextracellular antiproteases to below the level required to inhibitreleased proteases. Chlorinated oxidants and hydrogen peroxide caninactivate antiproteases such as alpha1-protease inhibitor andalpha2-macroglobulin, which are endogenous inhibitors of elastase, butsimultaneously activate latent metalloproteases such as collagenases andgelatinase, which contribute to the further inactivation ofantiproteases.

Cytoplasmic constituents of neutrophils may also be a cause of formationof specific anti-neutrophil cytoplasmic antibodies (ANCA), which areclosely related to the development of systemic vasculitis andglomerulonephritis. ANCA are antibodies directed against enzymes thatare found mainly within the azurophil or primary granules ofneutrophils. There are three types of ANCA that can be distinguished bythe patterns they produce by indirect immunofluorescence on normalethanol-fixed neutrophils. Diffuse fine granular cytoplasmicfluorescence (cANCA) is typically found in Wegener's granulomatosis, insome cases of microscopic polyarteritis and Churg Strauss syndrome, andin some cases of crescentic and segmental necrotizingglomerulonephritis. The target antigen is usually proteinase 3.Perinuclear fluorescence (pANCA) is found in many cases of microscopicpolyarteritis and glomerulonephritis. These antibodies are oftendirected against myeloperoxidase but other targets include elastase,cathepsin G, lactoferrin, lysozyme and beta-D-glucuronidase. The thirdgroup designated “atypical” ANCA includes neutrophil nuclearfluorescence and some unusual cytoplasmic patterns and while a few ofthe target antigens are shared with pANCA, the others have not beenidentified yet. pANCA are also found in a third of patients with Crohn'sdisease. The reported incidence of ANCA in rheumatoid arthritis and SLEvaries considerably but the patterns are predominantly pANCA andatypical ANCA.

The eosinophil is a terminally differentiated, end-stage leukocyte thatresides predominantly in submucosal tissue and is recruited to sites ofspecific immune reactions, including allergic diseases. The eosinophilcytoplasm contains large ellipsoid granules with an electron-densecrystalline nucleus and partially permeable matrix. In addition to theselarge primary crystalloid granules, there is another granule type thatis smaller (small granule) and lacks the crystalline nucleus. The largespecific granules of eosinophils contain at least four distinct cationicproteins, which exert a range of biological effects on host cells andmicrobial targets: major basic protein (MBP), eosinophil cationicprotein (ECP), eosinophil derived neurotoxin (EDN), and eosinophilperoxidase (EPO). Basophils contain about one fourth as much major basicprotein as eosinophils together with detectable amounts of EDN, ECP andEPO. Small amounts of EDN and ECP are also found in neutrophils (GleichG J. Mechanisms of eosinophil-associated inflammation. J Allergy ClinImmunol 2000; 105:651-663). MBP appears to lack enzymatic activity butis a highly cationic polypeptide which may exert its toxic activities byinteractions with lipid membranes leading to their derangement. Both MBPand EPO can act as selective allosteric inhibitors of agonist binding toM2 muscarinic receptors. These proteins may contribute to M2 receptordysfunction and enhance vagally mediated bronchoconstriction in asthma.EDN can specifically damage the myelin coat of neurons. Histaminase anda variety of hydrolytic lysosomal enzymes are also present in the largespecific granules of eosinophils. Among the enzymes in small granules ofeosinophils are aryl sulphatase, acid phosphatase, and a 92 kDametalloproteinase, a gelatinase. Eosinophils can elaborate cytokineswhich include those with potential autocrine growth-factor activitiesfor eosinophils and those with potential roles in acute and chronicinflammatory responses. Three cytokines have growth-factor activitiesfor eosinophils: granulocyte-macrophage colony-stimulating factor(GM-CSF), IL-3 and IL-5. Other cytokines produced by human eosinophilsthat may have activities in acute and chronic inflammatory responsesinclude IL-1-alpha, IL-6, IL-8, TNF-alpha and both transforming growthfactors, TGF-alpha and TGF-beta.

Eosinophils contain crystalloid granules that contain MBP, eosinophilcationic protein, EPO, and eosinophil-derived neurotoxin (Gleich, JAllergy Clin Immunol 2000; 105:651-663). The human promyelocytic cellline HL-60 clone 15 can be used to examine secretion of EPO. This cellline was established from a clone of HL-60 that had been grown at anelevated pH for two months (Fischkoff, Leuk Res 1988; 12:679-686) andthen treated with butyric acid to allow the cells to differentiate so asto exhibit many of the characteristics of peripheral blood eosinophils,including expression of eosinophil-specific granule proteins (Rosenberget al., J Exp Med 1989; 170:163-176; Tiffany et al., J Leukoc Biol 1995;58:49-54; Badewa et al., Exp Biol Med 2002; 227:645-651).

Eosinophils can participate in hypersensitivity reactions, especiallythrough two lipid inflammatory mediators, leukotriene C⁴ (LTC⁴) andplatelet activating factor (PAF). Both mediators contract airway smoothmuscle, promote the secretion of mucus, alter vascular permeability andelicit eosinophil and neutrophil infiltration. In addition to the directactivities of these eosinophil-derived mediators, MBP can stimulate therelease of histamine from basophils and mast cells, and MBP canstimulate the release of EPO from mast cells. Eosinophils can serve as alocal source of specific lipid mediators as well as induce the releaseof mediators from mast cells and basophils. Eosinophil granule contentis released following similar stimuli to neutrophil granules, e.g.during phagocytosis of opsonized particles and by chemotactic factors.Neutrophil lysosomal enzymes act primarily on material engulfed inphagolysosomes, while the eosinophil granule contents act mainly onextracellular target structure such as parasites and inflammatorymediators.

Monocyte and macrophage development takes place in the bone marrow andpasses through the following steps: stem cell; committed stem cell;monoblast; promonocyte; monocyte in bone marrow; monocyte in peripheralblood; and macrophage in tissues. Monocyte differentiation in the bonemarrow proceeds rapidly (1.5 to 3 days). During differentiation,granules are formed in monocyte cytoplasm and these can be divided as inneutrophils into at least two types. However, they are fewer and smallerthan their neutrophil counterparts (azurophil and specific granules).Their enzyme content is similar.

Granule-bound enzymes of monocytes/macrophages include lysozyme, acidphosphatase, and beta-glucuronidase. As a model for in vivo studies,lysozyme secretion from U937 cells was used. This cell line is derivedfrom a human histiocytic lymphoma and has been used as a monocytic cellline that can be activated by a variety of agonists, such as PMA (Hoffet al., J Leukoc Biol 1992; 52:173-182; Balboa et al., J Immunol 2003;170:5276-5280; Sundstrom et al., Int J Cancer 1976; 17:565-577).

Natural killer (NK) cells and cytotoxic lymphocytes contain potentcytotoxic granules including perform, a pore-forming protein, andgranzymes, lymphocyte-specific serine proteases. For example, the NK-92cell line is an IL-2-dependent human line established from a patientwith rapidly progressive non-Hodgkin's lymphoma (Gong J H., Maki G,Klingemann H G. Characterization of a human cell line (NK-92) withphenotypical and functional characteristics of activated natural killercells. Leukemia 1994; 8:652-658). NK-92 cells express high levels ofmolecules involved in the perforin-granzyme cytolytic pathway thattargets a wide range of malignant cells (Gong et al, vide infra, andMaki G, Klingemann H G, Martinson J A, Tam Y K. Factors regulating thecytotoxic activity of the human natural killer cell line, NK-92. JHematother Stem Cell Res 2001; 10:369-383).

Granzymes are exogenous serine proteases that are released bycytoplasmic granules within cytotoxic T cells and natural killer cells.Granzymes can induce apoptosis within virus-infected cells, thusdestroying them.

Extracellular release of a mediator of inflammation (inflammatorymediator) from a granulocyte (or leukocyte), and extracellular releaseof more than one mediator of inflammation (inflammatory mediator) from agranulocyte (or leukocyte) is sometimes referred to herein asdegranulation. In a preferred embodiment, the release of a mediator ofinflammation comprises release of said mediator from a granule locatedin the interior of a granulocyte or leukocyte. The release ofinflammatory mediator is preferably the release of an inflammatorymediator from these granules.

Neutrophils and macrophages, upon priming by pro-inflammatory agents(inflammatory stimulants) such as TNFα, dramatically increase theirsynthesis of MARCKS protein: as much as 90% of the new protein formed byneutrophils in response to either TNFα or lipopolysaccharide (LPS) isMARCKS (Thelen M, Rosen A, Nairn A C, Aderem A. Tumor necrosis factoralpha modifies agonist-dependent responses in human neutrophils byinducing the synthesis and myristoylation of a specific protein kinase Csubstrate. Proc Natl Acad Sci USA 1990; 87:5603-5607). MARCKS can thushave an important role in subsequent release of inflammatory mediatorswhen granule-containing cells, such as neutrophils and macrophages, arestimulated by agonists, especially those that work by activating PKC(Burgoyne et al., Physiol Rev 2003; 83:581-632; Logan et al. J AllergyClin Immunol 2003; 111: 923-932; Smolen et al., Biochim Biophys Acta1990; 1052:133-142; Niessen et al., Biochim. Biophys. Acta 1994;1223:267-273; Naucler et al., J Leukoc Biol 2002; 71:701-710).

In one aspect of this invention, administration of adegranulation-inhibiting amount of MANS peptide or an active fragmentthereof as described herein to a site of inflammation in a subject,which site of inflammation has resulted from the onset of entry of adisease, a condition, a trauma, a foreign body, or a combination thereofat the site of inflammation in the subject, can reduce the amount of amediator of inflammation released from infiltrating leukocytes at thesite of inflammation, where the leukocytes are preferably granulocytes.The administration of the MANS peptide and/or at least one activefragment thereof can reduce the amount of a mediator of inflammationreleased from leukocytes such as granulocytes infiltrating into the siteof inflammation. The degranulation-inhibiting amount of MANS peptide, orthe degranulation-inhibiting amount of an active fragment thereof, issufficient to reduce or inhibit the exocytotic release of inflammatorymediators from granules contained within the inflammatory cellsinfiltrating into the site. Degranulation-inhibiting efficacy ismeasured at a time after administration of the MANS peptide or thefragment thereof by comparison of the percent of inhibition (i.e.,percent of reduction) of the release of mediators of inflammation fromsaid cells (leukocytes or granulocytes or other inflammatory cells)relative to the level or amount or concentration of said mediators ofinflammation released or produced at approximately the same time in theabsence of MANS peptide and/or in the absence of the active fragmentthereof. Additionally, a skilled clinician can determine whetherinflammation at the tissue site has been reduced by measuring symptomsand parameters of inflammation known as indicators of the disease todetermine whether a sufficient or therapeutically effective amount MANSpeptide and/or an active fragment thereof has been administered. Asufficient degranulation-inhibiting amount is the percentage ofreduction of a mediator of inflammation released from a granulocyte, atthe site of inflammation, which is from about 1% to about 99%,preferably from 5% to about 99%, more preferably from about 10% to about99%, even more preferably from about 25% to 99%, and even morepreferably from about 50% to about 99% of the amount of said mediator ofinflammation released from said granulocyte in the absence of MANSpeptide or an active fragment thereof tested under the same conditions.

In one aspect of this invention, administration of adegranulation-inhibiting amount of MANS peptide to a site ofinflammatory stimulation in an animal, which site of inflammatorystimulation has been created by administration of aninflammation-stimulating amount of an inflammatory stimulant to saidsite, can reduce the amount of a mediator of inflammation released froma granulocyte, which granulocyte is stimulated by said inflammatorystimulant at said site of inflammatory stimulation, from about 1% toabout 99%, preferably from 5% to about 99%, more preferably from about10% to about 99%, even more preferably from about 25% to 99%, and evenmore preferably from about 50% to about 99% of the amount of saidmediator of inflammation released from said granulocyte in the absenceof MANS peptide in the presence of the identicalinflammation-stimulating amount of said inflammatory stimulant.

In another aspect of this invention, administration of adegranulation-inhibiting amount of MANS peptide to a site ofinflammatory stimulation in an animal, which site of inflammatorystimulation has been created by administration of aninflammation-stimulating amount of an inflammatory stimulant to saidsite, can reduce the amount of a mediator of inflammation released froma granulocyte, which granulocyte is stimulated by said inflammatorystimulant at said site of inflammatory stimulation, by 100% of theamount of said mediator of inflammation released from said granulocytein the absence of MANS peptide in the presence of the identicalinflammation-stimulating amount of said inflammatory stimulant.

An example of an inflammatory stimulant used in in vitro examples hereinis phorbol 12-myristate 13-acetate (PMA). Monocyte chemoattractantprotein (MCP-1) is nearly as effective as C5a, and much more potent thanIL-8, in the degranulation of basophils, resulting in histamine release.Histamine release can occur after stimulation with chemokines (i.e.,chemoattractant cytokines), RANTES and MIP-1.

In a preferred embodiment, relative to the basal concentration of MARCKSpeptide present at the site of inflammatory stimulation, thedegranulation-inhibiting amount of MANS peptide administered to a siteof inflammatory stimulation in an animal comprises from about 1 time toabout 1,000,000 times the concentration of the MARCKS peptide at saidsite of inflammatory stimulation, preferably from about 1 time to about100,000 times the concentration of the MARCKS peptide at said site ofinflammatory stimulation, more preferably from about 1 time to about10,000 times the concentration of the MARCKS peptide at said site ofinflammatory stimulation, even more preferably from about 1 time toabout 1,000 times the concentration of the MARCKS peptide at said siteof inflammatory stimulation, even more preferably from about 1 time toabout 100 times the concentration of the MARCKS peptide at said site ofinflammatory stimulation, and even more preferably from about 1 time toabout 10 times the concentration of the MARCKS peptide at said site ofinflammatory stimulation.

In a preferred embodiment, the granulocyte resides on or in the airwayof an animal, preferably a human, and the MANS peptide is administeredby inhalation, such as by inhalation of a pharmaceutical compositioncomprising the MANS peptide, for example a pharmaceutical compositioncomprising the MANS peptide and an aqueous solution, which compositionis administered in the form of an aerosol, or a pharmaceuticalcomposition comprising the MANS peptide in the form of a dry powder,which composition is administered using a dry powder inhaler. Othermethods and devices known in the art for administration of a solution orpowder by inhalation such as, for example, droplets, sprays, andnebulizers, can be useful.

In some embodiments, it is possible that the peptide of the presentinvention may block secretory processes that are physiologicallyimportant, including basal secretory functions. Although inventors donot wish to be bound to any particular theory of the invention, it isthought that the mechanisms regulating such basal secretion aredifferent than those regulating stimulated secretion. Alternatively,basal secretory mechanisms may require less MARCKS protein thanstimulated secretion. Basal secretion may be preserved since alltherapies to block MARCKS-mediated secretion may not eliminate allMARCKS function.

As used herein, the term “MARCKS nucleotide sequence” refers to anynucleotide sequence derived from a gene encoding a MARCKS protein,including, for example, DNA or RNA sequence, DNA sequence of the gene,any transcribed RNA sequence, RNA sequence of the pre-mRNA or mRNAtranscript, and DNA or RNA bound to protein.

Precise delivery of the MARCKS-blocking peptide may also overcome anypotential limitations of blocking important secretory processes.Delivering such agents to the respiratory tract should be readilyaccomplished with inhaled formulations. Since these agents may be usefulin treating inflammatory bowel disease, one can envision delivery of theblocking agents into the rectum/colon/intestinal tract via enema orsuppositories. Injections or transdermal delivery into inflamed jointsmay yield relief to patients with arthritic or autoimmune diseases bylimiting the secretion from localized inflammatory cells. Injection intoareas surrounding nerve endings may inhibit secretion of some types ofneurotransmitters, blocking transmission of severe pain or uncontrolledmuscle spasms. Delivery of the peptide for the treatment of inflammatoryskin diseases should be readily accomplished using various topicalformulations known in the art.

It has been shown that the myristoylated alanine-rich C kinase substrate(MARCKS), a widely distributed PKC substrate may be a key regulatorymolecule mediating mucin granule release by normal human bronchialepithelial (NHBE) cells. Secretion of mucin from these cells may bemaximized by activation of both PKC and PKG. It is believed that MARCKSserves as the point of convergence for coordinating the actions of thesetwo protein kinases to control mucin granule release. The mechanismappears to involve PKC-dependent phosphorylation of MARCKS, whichreleases MARCKS from the plasma membrane into the cytoplasm, where it isin turn dephosphorylated by a protein phosphatase 2A (PP2A) that isactivated by PKG. This dephosphorylation may allow MARCKS to regain itsmembrane-binding capability, enabling its attachment to membranes ofcytoplasmic mucin granules. In addition, MARCKS interacts with actin andmyosin in the cytoplasm and thus may be able to tether the granules tothe cellular contractile apparatus, thus, mediating subsequent granulemovement and exocytosis. Secretion of the inflammatory mediatory MPOfrom neutrophils may also be maximized by activation of both PKC and PKG(as illustrated in FIGS. 11-15). It is possible that MARCKS serves asthe point of convergence for coordinating actions of these two proteinkinases that control secretion from membrane-bound compartments ininflammatory cells (i.e. secretion of MPO from neutrophils).

The present invention demonstrates secretion of the inflammatorymediator MPO from canine or human neutrophils was enhanced by concurrentactivation of both PKC and PKG, while activation of either kinase alonewas insufficient to induce a maximal secretory response. An enhancedsecretory response to PMA alone was documented in NHBE cells (FIG. 1,column 4) and in neutrophils (FIG. 11), although the magnitude of theresponse was much less than that observed by others in a rat goblet-likecell line. See, Abdullah et al, supra. In addition, although it wasreported previously that a cGMP analogue could induce significant mucinsecretion from cultured guinea pig tracheal epithelial cells (Fischer etal., supra), it should be noted that this response did not reachsignificant levels until 8 h of exposure. A secretory response with sucha long lag period is unlikely to be a direct effect and probablyinvolves de novo protein synthesis as opposed to release of preformedand stored cytoplasmic granules. Nevertheless, the apparent synergisticeffect involving cooperative activation of both PKC and PKG may suggesta complex and stringent signaling mechanism mediating mucin secretionand/or inflammatory mediators. Applicants note that the pathwaydisclosed below was used to study inflammatory mediator release fromneutrophils and is likely the same pathway as that used to study gobletcell secretions.

As stated above, the present invention may be used in a pharmaceuticalformulation. In certain embodiments, the drug product is present in asolid pharmaceutical composition that may be suitable for oraladministration. A solid composition of matter according to the presentinvention may be formed and may be mixed with and/or diluted by anexcipient. The solid composition of matter also may be enclosed within acarrier, which may be, for example, in the form of a capsule, sachet,tablet, paper, or other container. When the excipient serves as adiluent, it may be a solid, semi-solid, or liquid material that acts asa vehicle, carrier, or medium for the composition of matter.

Various suitable excipients will be understood by those skilled in theart and may be found in the National Formulary, 19: 2404-2406 (2000),the disclosure of pages 2404 to 2406 being incorporated herein in theirentirety. Examples of suitable excipients include, but are not limitedto, starches, gum arabic, calcium silicate, microcrystalline cellulose,methacrylates, shellac, polyvinylpyrrolidone, cellulose, water, syrup,and methylcellulose. The drug product formulations additionally caninclude lubricating agents such as, for example, talc, magnesiumstearate and mineral oil; wetting agents; emulsifying and suspendingagents; preserving agents such as methyl- and propyl hydroxybenzoates;sweetening agents; or flavoring agents. Polyols, buffers, and inertfillers also may be used. Examples of polyols include, but are notlimited to, mannitol, sorbitol, xylitol, sucrose, maltose, glucose,lactose, dextrose, and the like. Suitable buffers include, but are notlimited to, phosphate, citrate, tartrate, succinate, and the like. Otherinert fillers that may be used include those that are known in the artand are useful in the manufacture of various dosage forms. If desired,the solid formulations may include other components such as bulkingagents and/or granulating agents, and the like. The drug products of theinvention may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art.

To form tablets for oral administration, the composition of matter ofthe present invention may be made by a direct compression process. Inthis process, the active drug ingredients may be mixed with a solid,pulverant carrier such as, for example, lactose, saccharose, sorbitol,mannitol, starch, amylopectin, cellulose derivatives or gelatin, andmixtures thereof, as well as with an antifriction agent such as, forexample, magnesium stearate, calcium stearate, and polyethylene glycolwaxes. The mixture may then be pressed into tablets using a machine withthe appropriate punches and dies to obtain the desired tablet size. Theoperating parameters of the machine may be selected by the skilledartisan. Alternatively, tablets for oral administration may be formed bya wet granulation process. Active drug ingredients may be mixed withexcipients and/or diluents. The solid substances may be ground or sievedto a desired particle size. A binding agent may be added to the drug.The binding agent may be suspended and homogenized in a suitablesolvent. The active ingredient and auxiliary agents also may be mixedwith the binding agent solution. The resulting dry mixture is moistenedwith the solution uniformly. The moistening typically causes theparticles to aggregate slightly, and the resulting mass is pressedthrough a stainless steel sieve having a desired size. The mixture isthen dried in controlled drying units for the determined length of timenecessary to achieve a desired particle size and consistency. Thegranules of the dried mixture are sieved to remove any powder. To thismixture, disintegrating, antifriction, and/or anti-adhesive agents maybe added. Finally, the mixture is pressed into tablets using a machinewith the appropriate punches and dies to obtain the desired tablet size.The operating parameters of the machine may be selected by the skilledartisan.

If coated tablets are desired, the above prepared core may be coatedwith a concentrated solution of sugar or cellulosic polymers, which maycontain gum arabic, gelatin, talc, titanium dioxide, or with a lacquerdissolved in a volatile organic solvent or a mixture of solvents. Tothis coating various dyes may be added in order to distinguish amongtablets with different active compounds or with different amounts of theactive compound present. In a particular embodiment, the activeingredient may be present in a core surrounded by one or more layersincluding enteric coating layers.

Soft gelatin capsules may be prepared in which capsules contain amixture of the active ingredient and vegetable oil. Hard gelatincapsules may contain granules of the active ingredient in combinationwith a solid, pulverulent carrier, such as, for example, lactose,saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin,cellulose derivatives, and/or gelatin.

Liquid preparations for oral administration may be prepared in the formof syrups or suspensions, e.g., solutions containing an activeingredient, sugar, and a mixture of ethanol, water, glycerol, andpropylene glycol. If desired, such liquid preparations may comprise oneor more of following: coloring agents, flavoring agents, and saccharin.Thickening agents such as carboxymethylcellulose also may be used.

In the event that the above pharmaceuticals are to be used forparenteral administration, such a formulation may comprise sterileaqueous injection solutions, non-aqueous injection solutions, or both,comprising the composition of matter of the present invention. Whenaqueous injection solutions are prepared, the composition of matter maybe present as a water soluble pharmaceutically acceptable salt.Parenteral preparations may contain anti-oxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient. Aqueous and non-aqueous sterilesuspensions may comprise suspending agents and thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampules and vials. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

The composition of matter also may be formulated such that it may besuitable for topical administration (e.g., skin cream). Theseformulations may contain various excipients known to those skilled inthe art. Suitable excipients may include, but are not limited to, cetylesters wax, cetyl alcohol, white wax, glyceryl monostearate, propyleneglycol, monostearate, methyl stearate, benzyl alcohol, sodium laurylsulfate, glycerin, mineral oil, water, carbomer, ethyl alcohol, acrylateadhesives, polyisobutylene adhesives, and silicone adhesives.

Having now described the invention, the same will be illustrated withreference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

EXAMPLES Mucin Hypersecretion from NHBE Cells Involves Activation ofBoth PKC and PKG

To determine the potential role of PKC and/or PKG in the mucin secretoryprocess, NHBE cells were exposed to the following two specific proteinkinase activators: the phorbol ester, phorbol 12-myristate 13-acetate(PMA), for activation of PKC, and the nonhydrolyzable cGMP analogue,8-Br-cGMP, for activation of PKG. Preliminary studies examining mucinsecretion in response to PMA stimulation at various concentrations fordifferent times (up to 1 μM for 2 h) indicated that activation of PKCalone did not induce significant mucin secretion from NHBE cells,although a moderate secretory response was repeatedly observed at PMAconcentrations higher than 100 nM (0.05<p<0.1). Also, the cells did notrespond to the cGMP analogues at concentrations as high as 500 μM for upto 2 h of exposure. However, a combination of PMA+8-Br-cGMP, affectingdual activation of PKC and PKG, provoked a rapid increase in secretion,approximately doubling it within 15 min of exposure (FIG. 1A). Thissecretory response induced by PMA+8-Br-cGMP was concentration-dependent,with maximal stimulation at 100 nM PMA+1 μM 8-Br-cGMP (FIGS. 1B and 1C).In FIGS. 1A, 1B and 1C, NHBE cells were exposed to indicated reagent(s)or medium alone (CTL) for 15 min. In FIG. 1D, NHBE cells werepreincubated with the indicated inhibitor for 15 min and then stimulatedwith 100 .mu.M UTP for 2 h. Secreted mucin in response to the treatmentwas collected and assayed by ELISA. Data are presented as mean.+−.S.E.(n=6 at each point). The * stands for significantly different frommedium control (p<0.05); # stands for different from medium control(0.05<p<0.1); and ‡.stands for significantly different from UTPstimulation CD<0.05).

UTP is a well defined pathophysiologically relevant mucin secretagogue.Lethem et al., Am. J. Respir. Cell Mol. Biol. 9, 315-322 (1993). Thepresent invention further demonstrates that UTP, at variousconcentrations, preferably 40 to 140 μM, may induce a significantincrease in mucin secretion from NHBE cells after a 2-h exposure. Todetermine whether PKC and PKG were involved in regulation of mucinsecretion in response to a pathophysiological stimulus, effects ofPKC/PKG inhibitors on UTP-induced mucin secretion were investigated.NHBE cells were preincubated with various inhibitors for 15 min and thenexposed to UTP (100 μM) plus the inhibitor for 2 h. The secreted mucinwas measured by ELISA. The results indicated that mucin secretionprovoked by UTP may require both PKC and PKG activities, as thesecretory response was attenuated independently by the PKC inhibitorcalphostin C (500 nM), the PKG inhibitor R_(p)-8-Br-PET-cGMP (10 μM), orthe soluble guanylyl cyclase (GC-S) inhibitor LY83583 (50 μM) but likelynot by the protein kinase A (PKA) inhibitor KT5720 (500 nM) (FIG. 1D).Apparently, mucin secretion in NHBE cells may be regulated by asignaling mechanism involving both PKC and PKG.

To address involvement of PKG in the secretory process, 8-Br-cGMP wasutilized in these studies. Although the primary physiological effect of8-Br-cGMP is to activate PKG, it also has been reported to act as anagonist for cGMP-gated ion channels in some cells and, at highconcentrations, to cross-activate PKA. To preclude the possibility thatcGMP-gated ion channels and/or PKA may play a role in mucin secretion byNHBE cells, R_(p)-8-Br-cGMP, a unique cGMP analogue that can activatecGMP-gated ion channels similar to 8-Br-cGMP but inhibit PKG activity,was used as an agonist to distinguish the effects of PKG and cGMP-gatedion channels on mucin release. As illustrated in the figures,particularly, FIG. 1A (column 11), R_(p)-8-Br-cGMP did not enhance mucinsecretion when added to the cells with PMA. Likewise, the specific PKAinhibitor, KT5720 (500 nM), did not affect mucin secretion induced byeither PMA+8-Br-cGMP or UTP (FIG. 1D, column 4). These studies maynegate the possibility that cGMP-gated ion channels or PKA areassociated with mucin secretion, indicating that activation of PKG inNHBE cells is the mechanism whereby 8-Br-cGMP contributes to enhancedsecretion. Furthermore, because UTP-induced mucin hypersecretion can beattenuated by the soluble guanylyl cyclase (GC-S) inhibitor LY83583, itis likely that activation of PKG occurs via the signaling pathway ofnitric oxide (NO)→GC-S→cGMP→PKG, as illustrated previously indifferentiated guinea pig tracheal epithelial cells in vitro.

Given the participation of both PKC and PKG in the mucin secretoryprocess, the present invention examines potential intracellularsubstrates of these enzymes that could play a role in signaling eventsdownstream of the kinase activation. Numerous intracellular substratescan be phosphorylated by PKC or PKG, and phosphorylation by PKC of onesuch substrate, MARCKS protein, seemed to be of particular interest.MARCKS phosphorylation has been observed to correlate with a number ofcellular processes involving PKC signaling and cytoskeletal contraction,such as cell movement, mitogenesis, and neural transmitter release.Because the dynamic process of secretion requires both kinase activationand translocation of intracellular granules to the cell periphery,MARCKS appeared to be a candidate for a mediator molecule connectingPKC/PKG activation and mucin granule exocytosis.

MARCKS is a Key Molecule Linking PKC/PKG Activation to Mucin Secretionin NHBE Cells

To address the signaling mechanism downstream of protein kinaseactivation, MARCKS protein, a specific cellular substrate of PKC thatmight play a role in linking kinase activation to granule release wasstudied. First, the presence of MARCKS in NHBE cells by [³H] myristicacid-labeled immunoprecipitation assay was confirmed. As illustrated inFIG. 2A, MARCKS was expressed in NHBE cells, and the majority of thisprotein was membrane-associated under unstimulated conditions. In FIG.2A, cells were labeled with [³H]myristic acid overnight and the membrane(lane 1) and the cytosol (lane 2) fractions were then isolated bydifferential centrifugation. A role for MARCKS as a key regulatorycomponent of the mucin secretory pathway may be demonstrated in threedifferent ways.

As stated above, direct involvement of MARCKS in mucin secretion by NHBEcells may be demonstrated by three separate lines of evidence. First,mucin secretion in response to stimulation by PMA+8-Br-cGMP or UTP wasinhibited in a concentration-dependent manner by the MANS peptide, whichhad the amino acid sequence identical to the N-terminal region ofMARCKS, whereas the corresponding control peptide (RNS), containing thesame amino acid composition but arranged in random order, did not affectsecretion. The N-terminal myristoylated domain of MARCKS is known tomediate the MARCKS-membrane association. As indicated in FIG. 8, MARCKSmay function as a molecular linker by interacting with granule membranesat its N-terminal domain and binding to actin filaments at its PSD site,thereby tethering granules to the contractile cytoskeleton for movementand exocytosis. FIG. 8 shows a possible mechanism depicting that mucinsecretagogue interacts with airway epithelial (goblet) cells andactivates two separate protein kinases, PKC and PKG. Activated PKCphosphorylates MARCKS, causing MARCKS translocation from the plasmamembrane to the cytoplasm, whereas PKG, activated via the nitric oxide(NO)→GC-S→cGMP→PKG pathway, in turn activates a cytoplasmic PP2A, whichdephosphorylates MARCKS. This dephosphorylation stabilizes MARCKSattachment to the granule membranes. In addition, MARCKS also interactswith actin and myosin, thereby linking granules to the cellularcontractile machinery for subsequent movement and exocytotic release.The attachment of MARCKS to the granules after it is released into thecytoplasm may also be guided by specific targeting proteins or someother forms of protein-protein interactions in which the N-terminaldomain of MARCKS is involved. In either case, the MANS peptide, or anactive fragment thereof, comprising at least 6 amino acids, would act toinhibit competitively targeting of MARCKS to the membranes of mucingranules, thereby blocking secretion.

A second test demonstrated the inhibitory effect of a MARCKS-specificantisense oligonucleotide on mucin secretion. As shown in FIGS. 3A-3C,the antisense oligonucleotide down-regulated MARCKS mRNA and proteinlevels in NHBE cells and substantially attenuated mucin secretioninduced by PKC/PKG activation. The inhibition was not as dramatic asthat seen with the MANS peptide, which might be due to the high levelsof endogenous MARCKS protein in NHBE cells and the relatively longhalf-life of MARCKS mRNA (t_(1/2)=4-6 h). In FIGS. 3A-3C, NHBE cellswere treated with the antisense or the control oligonucleotide for 3days and then stimulated with PMA (100 nM)+8-Br-cGMP (1 μM) for 15 min.Mucin secretion was analyzed by ELISA. Total RNA and protein wereisolated from treated cells. MARCKS mRNA was assessed by Northernhybridization, and protein was assessed by Western blot. In the PMA (100nM)+8-Br-cGMP (1 μM) FIG. 3A is a Northern blot that showed a decreaseof .about.15% in MARCKS mRNA compared with controls in the attachedchart; FIG. 3B is Western blot that showed a decrease of about 30% inMARCKS protein in the attached graph; and FIG. 3C shows mucinhypersecretion was attenuated significantly by the antisenseoligonucleotide, whereas the control oligonucleotide had no effect. Dataare presented as mean±S.E. (n=6 at each point) wherein the * issignificantly different from medium control (p<0.05); and the † issignificantly different from PMA+8-Br-cGMP stimulation (p<0.05).Additionally, it is noted that the term CTO is the controloligonucleotide, while the term ASO is an antisense oligonucleotide.

It has been demonstrated that antisense oligonucleotides that arecomplementary to specific RNAs can inhibit the expression of cellulargenes as proteins. See Erickson and Izant, Gene Regulation Biology OfAntisense RNA And DNA, Vol. 1, Raven Press, N.Y., 1992. For example,selective inhibition of a p21 gene that differed from a normal gene by asingle nucleotide has been reported. Chang et al., Biochemistry (1991),30:8283-8286. Many hypotheses have been proposed to explain themechanisms by which antisense oligonucleotides inhibit gene expression,however, the specific mechanism involved may depend on the cell typestudied, the RNA targeted, the specific site on the RNA targeted, andthe chemical nature of the oligonucleotide. Chiang et al., J. Biol.Chem. 1991, 266:18162-18171; Stein and Cohen, Cancer Res. 1988,48:2659-2668.

A third experiment indicated that transfection of HBE1 cells with aPSD-deleted mutant MARCKS resulted in significant repression of mucinsecretion induced by PKC/PKG activation. Deletion of the PSD wouldabolish the ability of MARCKS to bind to actin. As indicated in FIG. 8,by competing with native MARCKS for binding to granule membrane, thePSD-truncated MARCKS could thereby inhibit granule release as it isunable to interact with the actin filaments. Transfection of these cellswith the wild-type MARCKS cDNA did not further enhance mucin secretion.Western blot assay showed that the expression level of endogenous MARCKSin HBE1 cells was quite high, comparable with that in NHBE cells, andtransfection of wild-type MARCKS cDNA did not lead to notable increasesin overall MARCKS protein level in these cells. This may explain whytransfection with wild-type MARCKS did not further augment secretion andalso why transfection with the PSD-deleted MARCKS only partiallyhindered mucin secretion.

Peptide Blocking Studies—

NHBE cells were preincubated with either the MANS or the RNS peptide(1-100 .mu.M) for 15 min, and then PMA (100 nM)+8-Br-cGMP (1 μM) or UTP(100 μM) was added, and cells were incubated for an additional 15 min or2 h, respectively. Mucin secretion was measured by ELISA. As shown inFIG. 2B, incubation of NHBE cells with the MANS peptide resulted in aconcentration-dependent suppression of mucin secretion in response toPKC/PKG activation or UTP stimulation, whereas the control peptide (RNS)may not have affected secretion at these same concentrations. In FIG.2B, the MANS peptide blocks mucin hypersecretion induced byPMA+8-Br-cGMP or UTP in a concentration-dependent manner. NHBE cellswere preincubated with the indicated peptide for 15 min and then exposedto PMA (100 nM)+8-Br-cGMP (1 .mu.M) for 15 min or UTP (100 μM) for 2 h.Mucin secretion was measured by ELISA. Data are presented asmean.+−.S.E. (n=6 at each point), wherein * is significantly differentfrom medium control p<0.05); † is significantly different fromPMA+8-Br-cGMP stimulation (p<0.05); and ‡ is significantly differentfrom UTP stimulation (<0.05). Effects of the MANS peptide were likelynot related to cytotoxicity or general repression of cellular metabolicactivity, as neither the MANS nor the RNS peptide affected lactatedehydrogenase release or [³H]deoxyglucose uptake by the cells.

Antisense Oligonucleotide Studies—

To demonstrate further MARCKS as a key signaling component of the mucinsecretory pathway, the effect of an antisense oligonucleotide directedagainst MARCKS on mucin secretion was examined. As illustrated in FIG.3, this antisense oligonucleotide down-regulated both mRNA and proteinlevels of MARCKS in NHBE cells and significantly attenuated mucinsecretion induced by PMA+8-Br-cGMP, whereas a control oligonucleotidehad no effect.

MARCKS Serves as a Convergent Signaling Molecule Mediating Cross-Talk ofPKC and PKG Pathways

Collectively, the above results demonstrated that MARCKS was involvedintegrally in the mucin secretory process. Next the present inventorsaddressed how MARCKS acts as a key regulatory molecule upon which PKCand PKG converge to regulate mucin secretion. As illustrated in FIG. 5,MARCKS was phosphorylated by PKC and consequently translocated from themembrane to the cytoplasm. Here, PKG appeared to inducedephosphorylation of MARCKS (FIG. 5A, lane 4, and FIG. 5B). Thisdephosphorylation was reversed by the PKG inhibitor R_(p)-8-Br-PET-cGMP(FIG. 5A, lane 5), indicating the dephosphorylation was specificallyPKG-dependent. In FIG. 5, the NHBE cells were labeled with[³²P]orthophosphate and then exposed to the indicated reagents. MARCKSphosphorylation in response to the treatments was evaluated byimmunoprecipitation assay. In FIG. 5A, 8-Br-cGMP reversed MARCKSphosphorylation induced by PMA, and this effect of 8-Br-cGMP could beblocked by R_(p)-8-Br-PET-cGMP (PKG inhibitor) or okadaic acid (PP1/2Ainhibitor). For FIG. 5B, PMA-induced phosphorylation of MARCKS wasreversed by subsequent exposure of cells to 8-Br-cGMP. Lane 1, mediumalone for 8 min; lane 2, 100 nM PMA for 3 min; lane 3, 100 nM PMA for 3min and then with 1 μM 8-Br-cGMP for 5 min; lane 4, 100 nM PMA for 8min; lane 5, medium alone for 3 min and then 100 nM PMA+1 μM 8-Br-cGMPfor 5 min. In FIG. 5C, 8-Br-cGMP-induced MARCKS dephosphorylation wasattenuated by fostriecin in a concentration-dependent manner.

It is believed that PKG acts to dephosphorylate MARCKS via activation ofa protein phosphatase. As illustrated in FIG. 5A (lane 6), okadaic acidat 500 nM, a concentration that could inhibit both PP1 and PP2A, blockedPKG-induced dephosphorylation of MARCKS, suggesting that PKG causeddephosphorylation by activating PP1 and/or PP2A. Further studies withfostriecin and direct assay of phosphatase activities indicated thatonly PP2A was activated by PKG and was responsible for removal of thephosphate groups from MARCKS (FIG. 5C). It is likely that either okadaicacid or fostriecin, at concentrations that inhibited PKG-induceddephosphorylation of MARCKS, attenuated mucin secretion induced byPMA+8-Br-cGMP or UTP as exhibited in FIG. 6. FIG. 6 helps to demonstratethat PP2A is an essential component of the mucin secretory pathway. NHBEcells were preincubated with the indicated concentration of fostriecin,okadaic acid (500 nM), or medium alone for 15 min and then stimulatedwith PMA (100 nM)+8-Br-cGMP (1 μM) for 15 min or with UTP (100 μM) for 2h. Secreted mucin was measured by ELISA. Data are presented asmean.+−.S.E. (n=6 at each point) wherein * stands for significantlydifferent from medium control (p<0.05); † stands for significantlydifferent from PMA+8-Br-cGMP stimulation (p<0.05); and ‡ stands forsignificantly different from UTP stimulation p<0.05). Thus,dephosphorylation of MARCKS by a PKG-activated PP2A appears to be anessential component of the signaling pathway leading to mucin granuleexocytosis.

To reveal molecular events by which MARCKS links kinase activation tomucin secretion, phosphorylation of MARCKS in response to PKC/PKGactivation was investigated in depth. As illustrated in FIG. 4A, PMA(100 nM) likely induced a significant increase (3-4-fold) in MARCKSphosphorylation in NHBE cells, and this phosphorylation was attenuatedby the PKC inhibitor calphostin C (500 nM). Once phosphorylated, MARCKSwas translocated from the plasma membrane to the cytoplasm (FIG. 4B).More specifically, FIG. 4A shows the activation of PKC results in MARCKSphosphorylation in NHBE cells. Cells were labeled with[³²P]orthophosphate for 2 h and then exposed to the stimulatory and/orinhibitory reagents. MARCKS phosphorylation in response to thetreatments was evaluated by immunoprecipitation as described. Lane 1,medium control; lane 2 the vehicle, 0.1% Me.sub.2SO; lane 3, 100 nM4α-PMA; lane 4, 100 nM PMA; lane 5, 100 nM PMA+500 nM calphostin C; lane6, 500 nM calphostin C. FIG. 4B demonstrates phosphorylated MARCKS istranslocated from the plasma membrane to the cytoplasm. ³²P-Labeledcells were exposed to PMA (100 nM) or medium alone for 5 min, and thenthe membrane and the cytosol fractions were isolated. Activation of PKGby 8-Br-cGMP (1 μM, another kinase activation event necessary forprovoking mucin secretion, did not lead to MARCKS phosphorylation, but,in fact, the opposite effect was observed: MARCKS phosphorylationinduced by PMA was reversed by 8-Br-cGMP (FIG. 5A). This effect of8-Br-cGMP was not due to suppression of PKC activity, as the PMA-inducedphosphorylation could be reversed by subsequent addition of 8-Br-cGMP tothe cells (FIG. 5B). Therefore, PKG activation likely results indephosphorylation of MARCKS.

Further investigation demonstrated that PKG-induced MARCKSdephosphorylation was blocked by 500 nM okadaic acid, a proteinphosphatase (type 1 and/or 2A (PP1/2A)) inhibitor (FIG. 5A, lane 6).Thus, it appeared that the dephosphorylation was mediated by PP1 and/orPP2A. To define the subtype of protein phosphatase involved, a novel andmore specific inhibitor of PP2A, fostriecin (IC₅₀=3.2 nM), was utilizedin additional phosphorylation studies. As illustrated in FIG. 5C,fostriecin inhibited PKG-induced MARCKS dephosphorylation in aconcentration-dependent manner (1-500 nM), suggesting that PKG inducedthe dephosphorylation via activation of PP2A. To confirm furtheractivation of PP2A by PKG in NHBE cells, cytosolic PP1 and PP2Aactivities were determined after exposure of the cells to 8-Br-cGMP.PP2A activity was increased approximately 3-fold (from 0.1 to 0.3nmol/min/mg proteins, p<0.01) at concentrations of 8-Br-cGMP as low as0.1 .mu.M, whereas PP1 activity remained unchanged. This data indicatesthat PP2A may be activated by PKG and is responsible for thedephosphorylation of MARCKS. Accordingly, this PP2A activity appearedcritical for mucin secretion to occur; when PKG-induced MARCKSdephosphorylation was blocked by okadaic acid or fostriecin, thesecretory response to PKC/PKG activation or UTP stimulation wasameliorated (FIG. 6).

MARCKS Associates with Actin and Myosin in the Cytoplasm

FIG. 7 depicts a radiolabeled immunoprecipitation assay which revealsthat MARCKS may associate with two other proteins (.about 0.200 and.about 0.40 kDa) in the cytoplasm. In FIG. 7 NHBE cells were labeledwith [³H]leucine and [³H]proline overnight, and the membrane and thecytosol fractions were prepared as described under “ExperimentalProcedures.” Isolated fractions were precleared with the nonimmunecontrol antibody (6F6). The cytosol was then divided equally into twofractions and used for immunoprecipitation carried out in the presenceof 10 μM cytochalasin D (Biomol, Plymouth Meeting, Pa.) with theanti-MARCKS antibody 2F12 (lane 2) and the nonimmune control antibody6F6 (lane 3), respectively. MARCKS protein in the membrane fraction wasalso assessed by immunoprecipitation using the antibody 2F12 (lane 1).The precipitated protein complex was resolved by 8% SDS-polyacrylamidegel electrophoresis and visualized by enhanced autoradiography. MARCKSappeared to associate with two cytoplasmic proteins with molecularmasses of .about 200 and .about 40 kDa, respectively. These twoMARCKS-associated proteins were excised from the gel and analyzed bymatrix-assisted laser desorption ionization/time of flight massspectrometry/internal sequencing (the Protein/DNA Technology Center ofRockefeller University, N.Y.). The obtained peptide mass and sequencedata were used to search protein databases via Internet programsProFound and MS-Fit. Results indicate that they are myosin (heavy chain,non-muscle type A) and actin, respectively. Matrix-assisted laserdesorption ionization/time of flight mass spectrometry/internal sequenceanalysis indicates that these two MARCKS-associated proteins were myosin(heavy chain, non-muscle type A) and actin, respectively.

These studies suggest a new paradigm for the signaling mechanismcontrolling exocytotic secretion of airway mucin granules as well asproviding what is believed to be the first direct evidence demonstratinga specific biological function of MARCKS in a physiological process.MARCKS serves as a key mediator molecule regulating mucin granulerelease in human airway epithelial cells. It is believed thatelicitation of airway mucin secretion requires dual activation andsynergistic actions of PKC and PKG. Activated PKC phosphorylates MARCKS,resulting in translocation of MARCKS from the inner face of the plasmamembrane into the cytoplasm. Activation of PKG in turn activates PP2A,which dephosphorylates MARCKS in the cytoplasm. Because the membraneassociation ability of MARCKS is dependent on its phosphorylation statethis dephosphorylation may allow MARCKS to regain its membrane-bindingcapability and may enable MARCKS to attach to membranes of cytoplasmicmucin granules. By also interacting with actin and myosin in thecytoplasm (FIG. 7), MARCKS may then be able to tether granules to thecellular contractile apparatus, mediating granule movement to the cellperiphery and subsequent exocytotic release. The wide distribution ofMARCKS suggests the possibility that this or a similar mechanism mayregulate secretion of membrane-bound granules in various cell typesunder normal or pathological conditions.

The invention also relates to a new method for blocking any cellularexocytotic secretory process, especially those releasing inflammatorymediators from granules contained within inflammatory cells, whosestimulatory pathways involve the protein kinase C (PKC) substrate MARCKSprotein and release of contents from membrane-bound vesicles.Specifically, the inventors have shown that stimulated release of theinflammatory mediator myloperoxidase from human (FIG. 9) or canine (FIG.10) neutrophils can be blocked in a concentration-dependent manner bythe MANS peptide. Specifically, FIG. 9 shows isolated neutrophils thatwere stimulated to secrete myloperoxidase (MPO) with 100 nM PMA and 10.mu.M 8-Br-cGMP. 100 μM MANS peptide decreased secretion of MPO tocontrol levels (*=p<0.05). 10 μM MANS causes a slight decrease in MPOsecretion. 10 or 100 μM of a control peptide (RNS) has no effect on MPOsecretion. In FIG. 10, isolated neutrophils were stimulated to secretemyloperoxidase (MPO) with 100 nM PMA and 10 μM 8-Br-cGMP. 100 μM MANSpeptide decreased secretion of MPO to control levels (*=p<0.05). 10 μMMANS causes a slight decrease in MPO secretion. 10 or 100 μM of acontrol peptide (RNS) has no effect on MPO secretion. Thus, the peptidemay be used therapeutically to block the release of mediators ofinflammation secreted from infiltrating inflammatory cells in anytissues. Many of these released mediators are responsible for theextensive tissue damage observed in a variety of chronic inflammatorydiseases (i.e., respiratory diseases such as asthma, chronic bronchitisand COPD, inflammatory bowel diseases including ulcerative colitis andCrohn's disease, autoimmune diseases, skin diseases such as rosacea,eczema; and severe acne, arthritic and pain syndromes such as rheumatoidarthritis and fibromyalgia). This invention may be useful for treatingdiseases such as arthritis, chronic bronchitis, COPD and cysticfibrosis. This invention is accordingly useful for the treatment in bothhuman and animal diseases, especially those affecting equines, canines,felines, and other household pets.

FIGS. 11-15 show MPO secretion for both humans and canines. In all ofthese experiments, isolated neutrophils were stimulated with LPS at aconcentration of 1×10⁻⁶ M for 10 minutes at 37° C. prior to adding thestimuli as indicated in the figures. The LPS primes the cells so theycan respond to a secretagogue.

In one embodiment, this invention discloses a method of regulating aninflammation in a subject comprising administering a therapeuticallyeffective amount of a pharmaceutical composition comprising a MANSpeptide or an active fragment thereof. In one aspect of this embodiment,said active fragment of the MANS protein comprises at least six aminoacids. In another aspect, said inflammation is caused by respiratorydiseases, bowel diseases, skin diseases, autoimmune diseases and painsyndromes. In another aspect, said respiratory diseases are selectedfrom the group consisting of asthma, chronic bronchitis, and COPD. Inanother aspect, said bowel diseases are selected from the groupconsisting of ulcerative colitis, Crohn's disease and irritable bowelsyndrome. In another aspect, said skin diseases are selected from thegroup consisting of rosacea, eczema, psoriasis and severe acne. Inanother aspect, said inflammation is caused by arthritis or cysticfibrosis. In another aspect, said subject is a mammal. Additionally, inanother aspect, said mammal is selected from the group consisting ofhumans, canines, equines and felines. In another aspect, saidadministering step is selected from the group consisting of topicaladministration, parenteral administration, rectal administration,pulmonary administration, nasal administration, inhalation and oraladministration. In another aspect, said pulmonary administration isselected from the group of aerosol, dry powder inhaler, metered doseinhaler, and nebulizer.

In another embodiment, this invention discloses a method for regulatinga cellular secretory process in a subject comprising administering atherapeutically effective amount of a pharmaceutical compositioncomprising at least one compound comprising a MANS peptide or an activefragment thereof, that regulates an inflammatory mediator in a subject.In one aspect of this embodiment, said active fragment of the MANSprotein comprises at least six amino acids. In another aspect, saidregulating a cellular secretory process is blocking or reducing acellular secretory process. In another aspect, said inflammatorymediator is caused by respiratory diseases, bowel diseases, skindiseases, autoimmune diseases and pain syndromes. In another aspect,said respiratory diseases are selected from the group consisting ofasthma, chronic bronchitis, and COPD. In another aspect, said boweldiseases are selected from the group consisting of ulcerative colitis,Crohn's disease and irritable bowel syndrome. In another aspect, saidskin diseases are selected from the group consisting of rosacea, eczema,psoriasis and severe acne. In another aspect, said inflammatory mediatoris caused by arthritis or cystic fibrosis. In another aspect, saidsubject is a mammal. In another aspect, said mammal is selected from thegroup consisting of humans, canines, equines and felines. In anotheraspect, said administering step is selected from the group consisting oftopical administration, parenteral administration, rectaladministration, pulmonary administration, nasal administration,inhalation and oral administration. In another aspect, said pulmonaryadministration is selected from the group of aerosol, dry powderinhaler, metered dose inhaler, and nebulizer.

In another embodiment, this invention discloses a method of reducinginflammation in a subject comprising administering a therapeuticallyeffective amount of a compound that inhibits the MARCKS-related releaseof inflammatory mediators, whereby the release of inflammatory mediatorsin the subject is reduced compared to that which would occur in theabsence of said treatment. In one aspect of this embodiment, saidcompound is at least one active fragment of a MARCKS protein. In anotheraspect, said active fragment is at least six amino acids in length. Inanother aspect, said compound is a MANS peptide or an active fragmentthereof. In another aspect, said compound is an antisenseoligonucleotide directed against the coding sequence of a MARCKS proteinor an active fragment thereof. In another aspect, said active fragmentis at least six amino acids in length.

In another embodiment, this invention discloses a method of reducinginflammation in a subject comprising administering a therapeuticallyeffective amount of a pharmaceutically active composition comprising acompound that inhibits the MARCKS-related release of inflammatorymediators, whereby the inflammation in the subject is reduced comparedto that which would occur in the absence of said treatment. In oneaspect of this embodiment, said compound is an active fragment of aMARCKS protein. In another aspect, said active fragment is at least sixamino acids in length. In another aspect, said compound is a MANSpeptide or an active fragment thereof. In another aspect, said compoundis an antisense oligonucleotide directed against the coding sequence ofa MARCKS protein or an active fragment thereof. In another aspect, saidactive fragment is at least six amino acids in length. The presentinvention is intended to encompass a composition that contains one ormore of the MANS peptide or its active fragments and use thereof in thetreatment of inhibiting the release of inflammatory mediators fromgranules or vesicles of inflammatory cells.

In another embodiment, this invention discloses a method of regulatingmucin granule release in a subject comprising administering a compoundthat regulates mucin granule release, whereby mucin granules are reducedas compared to that which would occur in the absence of said mucingranules. In one aspect of this embodiment, said compound is an activefragment of a MARCKS protein. In another aspect, said compound is a MANSpeptide.

In another embodiment, this invention discloses a method of regulatingexocytotic secretion of airway mucin granules in a subject comprising:administering a compound that regulates mucin granule release, wherebymucin granules are reduced as compared to that which would occur in theabsence of said mucin granules. In one aspect of this embodiment, saidcompound is an active fragment of a MARCKS protein. In another aspect,said compound is a MANS peptide.

In another embodiment, this invention discloses a method of modulatingmucus secretion in a subject comprising: administering a therapeuticamount of an antisense sequence that are complementary to sequencesencoding a MARCKS protein or an active fragment thereof, wherein mucussecretion by said cell is inhibited compared to that which would occurin the absence of such administration. In one aspect of this embodiment,said sequence is at least eighteen nucleic acids in length. In anotheraspect, said compound is complementary to sequences encoding a MANSpeptide or an active fragment thereof. In another aspect, saidmodulating mucus secretion is blocking or reducing mucus secretion.

In another embodiment, this invention discloses a method of reducing orinhibiting inflammation in a subject comprising administering atherapeutically effective amount of at least one peptide comprising MANSpeptide or an active fragment thereof effective to modulate aninflammatory mediator at the inflammation site. In one aspect of thisembodiment, said active fragment is at least six amino acids in length.In another aspect, said inflammatory mediators are produced by cellsselected from the group consisting of neutrophils, basophils,eosinophils, monocytes and leukocytes. Preferably the cells areleukocytes, more preferably granulocytes, and even more preferablyneutrophils, basophils, eosinophils or a combination thereof. In anotheraspect, the agent is administered orally, parenterally, cavitarily,rectally or through an air passage. In another aspect, said compositionfurther comprises a second molecule selected from the group consistingof an antibiotic, an antiviral compound, an antiparasitic compound, ananti-inflammatory compound, and an immunosuppressant.

An active fragment of a MANS peptide can be selected from the groupconsisting of the myristoylated peptides of SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQID NO 19.

In another aspect of this invention, the methods disclosed in thisinvention can be accomplished by use of or administering of combinationsof the peptides disclosed in the invention, i.e., by use of oradministering of a peptide selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO 19, and combinations thereof.Preferably a single peptide is used or administered in the methodsdisclosed herein.

In response to protein kinase C (PKC) activation by an inflammatorystimulant, degranulation in a cell selected from the group consisting ofneutrophils, eosinophils, monocytes/macrophages and lymphocytes can beattenuated by pre-incubation and by co-incubation with a peptideidentical to the N-terminal region of MARCKS protein, wherein thepeptide is selected from the group consisting of the MANS peptide (SEQID NO: 1) and myristoylated N-terminal fragments thereof (SEQ ID NO: 3to 19). Although time courses and concentrations can vary among celltypes, in all cases the MANS peptide attenuates PKC-induceddegranulation.

Methods and Materials

NHBE Cell Culture—

Expansion, cryopreservation, and culture of NHBE cells in the air/liquidinterface were performed as described previously. See, Krunkosky et al.Briefly, NHBE cells (Clonetics, San Diego, Calif.) were seeded in ventedT75 tissue culture flasks (500 cells/cm²) and cultured until cellsreached 75-80% confluence. Cells were then dissociated by trypsin/EDTAand frozen as passage-2. Air/liquid interface culture was initiated byseeding passage-2 cells (2×10⁴ cells/cm²) in TRANSWELL® clear cultureinserts (Costar, Cambridge, Mass.) that were thinly coated with rat tailcollagen, type I (Collaborative Biomedical, Bedford, Mass.). Cells werecultured submerged in medium in a humidified 95% air, 5% CO₂ environmentfor 5-7 days until nearly confluent. At that time, the air/liquidinterface was created by removing the apical medium and feeding cellsbasalaterally. Medium was renewed daily thereafter. Cells were culturedfor an additional 14 days to allow for full differentiation.

Measurement of Mucin Secretion by ELISA—

Before collection of “base line” and “test” mucin samples, theaccumulated mucus at the apical surface of the cells was removed bywashing with phosphate-buffered saline, pH 7.2. To collect the base-linesecretion, cells were incubated with medium alone, and secreted mucin inthe apical medium was collected and reserved. Cells were rested for 24 hand then exposed to medium containing the selected stimulatory and/orinhibitory reagents (or appropriate controls), after which secretedmucin was collected and reserved as the test sample. Incubation timesfor the base line and the test were the same but varied depending on thetest reagent utilized. Both base line and test secretions were analyzedby ELISA using an antibody capture method as known in the art. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, pp. 570-573, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988). The primaryantibody for this assay was 17Q2 (Babco, Richmond, Calif.), a monoclonalantibody that reacts specifically with a carbohydrate epitope on humanairway mucins. The ratio of test/base-line mucin, is similar to a“secretory index”, was used to quantify mucin secretion, allowing eachculture dish to serve as its own control and thus, minimizing deviationcaused by variability among culture wells. Wright et al., Am. J.Physiol. 271, L854-L861 (1996). Levels of mucin secretion were reportedas percentage of the medium control.

Radiolabeled Immunoprecipitation Assay—

When labeling with [³²P]phosphate, cells were preincubated for 2 h inphosphate-free Dulbecco's modified Eagle's medium containing 0.2% bovineserum albumin and then labeled with 0.1 mCi/ml [³²P]orthophosphate (9000Ci/mmol, PerkinElmer Life Sciences) for 2 h. For labeling with[³H]myristic acid or ³H -amino acids, cells were incubated overnight inmedium containing 50 μCi/ml [³H]myristic acid (49 Ci/mmol, PerkinElmerLife Sciences) or 0.2 mCi/ml [³H]leucine (159 Ci/mmol, PerkinElmer LifeSciences) plus 0.4 mCi/ml [³H]proline (100 Ci/mmol, PerkinElmer LifeSciences). Following labeling, cells were exposed to stimulatoryreagents for 5 mM. When an inhibitor was used, cells were preincubatedwith the inhibitor for 15 min prior to stimulation. At the end of thetreatments, cells were lysed in a buffer containing 50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1 mMphenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 μg/ml pepstatin A,and 10 μg/ml leupeptin. Trichloroacetic acid precipitation andscintillation counting may determine the radiolabeling efficiency ineach culture. Immunoprecipitation of MARCKS protein was carried outaccording to the method of Spizz and Blackshear using cell lysatescontaining equal counts/min. Spizz et al., J. Biol. Chem. 271, 553-562(1996). Precipitated proteins were resolved by 8% SDS-polyacrylamide gelelectrophoresis and visualized by autoradiography. Anti-human MARCKSantibody (2F12) and nonimmune control antibody (6F6) were used in thisassay.

To assess MARCKS or MARCKS-associated protein complexes in differentsubcellular fractions, radiolabeled and treated cells were scraped intoa homogenization buffer (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM EDTA,1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 μg/ml pepstatinA, 10 μg/ml leupeptin) and then disrupted by nitrogen cavitation (800pounds/square inch for 20 min at 4° C.). Cell lysates were centrifugedat 600×g for 10 min at 4° C. to remove nuclei and unbroken cells.Post-nuclear supernatants were separated into membrane and cytosolfractions by ultracentrifugation at 400,000×g for 30 min at 4° C. Themembrane pellet was solubilized in the lysis buffer by sonication.Immunoprecipitation was then carried out as described above.

MARCKS-Related Peptides—

Both the myristoylated N-terminal sequence (MANS) and the randomN-terminal sequence (RNS) peptides were synthesized at GenemedSynthesis, Inc. (San Francisco, Calif.), then purified by high pressureliquid chromatography (>95% pure), and confirmed by mass spectroscopywith each showing one single peak with an appropriate molecular mass.The MANS peptide consisted of sequence identical to the first 24 aminoacids of MARCKS, i.e. the myristoylated N-terminal region that mediatesMARCKS insertion into membranes, MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1 (where MA=N-terminal myristate chain). The corresponding controlpeptide (RNS) contained the same amino acid composition as the MANS butarranged in random order, MA-GTAPAAEGAGAEVKRASAEAKQAF (SEQ ID NO: 2).The presence of the hydrophobic myristate moiety in these syntheticpeptides enhances their permeability to the plasma membranes, enablingthe peptides to be taken up readily by cells. To determine the effectsof these peptides on mucin secretion, cells were preincubated with thepeptides for 15 min prior to addition of secretagogues, and mucinsecretion was then measured by ELISA.

Antisense Oligonucleotides—

MARCKS antisense oligonucleotide and its corresponding controloligonucleotide were synthesized at Biognostik GmbH (Gottingen,Germany). NHBE cells were treated with 5 μM antisense or controloligonucleotide apically for 3 days (in the presence of 2 μg/mllipofectin for the first 24 h). Cells were then incubated withsecretagogues, and mucin secretion was measured by ELISA. Total RNA andprotein were isolated from treated cells. MARCKS mRNA was assessed byNorthern hybridization according to conventional procedures using humanMARCKS cDNA as a probe. MARCKS protein level was determined by Westernblot using purified anti-MARCKS IgG1 (clone 2F12) as the primarydetection antibody.

Transient Transfection—

The phosphorylation site domain (PSD) of MARCKS contains thePKC-dependent phosphorylation sites and the actin filament-binding site.To construct a PSD-deleted MARCKS cDNA, two fragments flanking the PSDsequence (coding for 25 amino acids) were generated by polymerase chainreaction and then ligated through the XhoI site that was attached to the5′-ends of oligonucleotide primers designed for the polymerase chainreaction. The resultant mutant cDNA and the wild-type MARCKS cDNA wereeach inserted into a mammalian expression vector pcDNA4/TO (Invitrogen,Carlsbad, Calif.). Isolated recombinant constructs were confirmed byrestriction digests and DNA sequencing.

HBE1 is a papilloma virus-transformed human bronchial epithelial cellline capable of mucin secretion when cultured in air/liquid interface.Transfection of HBE1 cells was carried out using the Effectenetransfection reagent (Qiagen, Valencia, Calif.) according to themanufacturer's instructions. Briefly, differentiated HBE1 cells grown inair/liquid interface were dissociated by trypsin/EDTA and re-seeded in12-well culture plates at 1×10⁵ cells/cm². After overnight incubation,cells were transfected with the wild-type MARCKS cDNA, the PSD-truncatedMARCKS cDNA, or vector DNA. Cells were cultured for 48 h to allow geneexpression and then exposed to secretagogues and mucin secretionmeasured by ELISA. All transfections were carried out in the presence ofpcDNA4/TO/lacZ plasmid (Invitrogen) (DNA ratio 6:1, total 1 μg DNA,ratio of DNA to Effectene reagent=1:25) to monitor variations intransfection efficiency. Results showed no significant difference in.beta.-galactosidase activities in cell lysates isolated from thetransfected cells, indicating similar transfection efficiency amongdifferent DNA constructs (data not shown).

Protein Phosphatase Activity Assay—

PP1 and PP2A activities were measured using a protein phosphatase assaysystem (Life Technologies, Inc.) as known in the art with slightmodification. Huang et al., Adv. Exp. Med. Biol. 396, 209-215 (1996).Briefly, NHBE cells were treated with 8-Br-cGMP or medium alone for 5min. Cells were then scraped into a lysis buffer (50 mM Tris-HCl (pH7.4), 0.1% .beta.-mecaptoethanol, 0.1 mM EDTA, 1 mM benaamidine, 10μg/ml pepstatin A, 10 μg/ml leupeptin) and disrupted by sonication for20 s at 4° C. Cell lysates were centrifuged and the supernatants savedfor phosphatase activity assay. The assay was performed using³²P-labeled phosphorylase A as a substrate. Released ³²P_(i) was countedby scintillation. The protein concentration of each sample wasdetermined by the Bradford assay. PP2A activity was expressed as thesample total phosphatase activity minus the activity remaining in thepresence of 1 nM okadaic acid. PP1 activity was expressed as thedifference between the activities remaining in the presence of 1 nM and1 μM okadaic acid, respectively. Protein phosphatase activities werereported as nmol of P_(i) released per min/mg total protein.

Cytotoxicity Assay—

All reagents used in treating NHBE cells were examined for cytotoxicityby measuring the total release of lactate dehydrogenase from the cells.The assay was carried out using the Promega Cytotox 96 Kit according tothe manufacturer's instructions. All experiments were performed withreagents at non-cytotoxic concentrations.

Statistical Analysis—

Data were analyzed for significance using one-way analysis of variancewith Bonferroni post-test corrections. Differences between treatmentswere considered significant at p<0.05.

Isolation of PMNs from Canine Blood—

The steps involved in isolating PMN include collecting 10 ml ACDanticoagulated blood. Then layering 5 ml on 3.5 ml PMN isolation mediawhile ensuring that the PMN isolation media (IM) was at room temperature(RI). Next, the blood was centrifuged at room temperature for 30′, 550×gat 1700 RPMs. The low lower white band was transferred into 15 mlconical centrifuge tube (CCFT). Next, 2V HESS with 10% fetal bovineserum (PBS) was added and centrifuged at room temperature for 10′, 400×gat 1400 RPMs. The pellet was then resuspended in 5 ml 1-LESS with PBS.The cell suspension was added to 50 ml CCFT containing 20 ml of ice cold0.88% NH₄Cl and inverted two to three times. The resulting product wascentrifuged for 10′, 800×g at 2000 RPMs, then aspirated and resuspendedin 5 ml HBSS with FBS. The prep was examined by counting and cytospinand preferably for whole blood, the cell number should be between10⁹-10¹¹ cells and for PMNs, cell number should be between 2-4×10⁷cells. See generally, Wang et al., J. Immunol., “Neutrophil-inducedchanges in the biomechanical properties of endothelial cells: roles ofICAM-1 and reactive oxygen species,” 6487-94 (2000).

MPO Colorimetric Enzyme Assay—

Samples were assayed for MPO activity in 96 well round bottom microtiterplates using a sandwich ELISA kit (R & D Systems, Minneapolis, Minn.).Briefly, 20 microliters of sample is mixed with 180 microliters ofsubstrate mixture containing 33 mM potassium phosphate, pH 6.0, 0.56%Triton X-100, 0.11 mM hydrogen peroxide, and 0.36 mM O-DiannisidineDihydrochloride in an individual microtiter well. The finalconcentrations in the assay mixture are: 30 mM potassium phosphate, pH6.0, 0.05% Triton X-100, 0.1 mM hydrogen peroxide, and 0.32 mMO-Diannisidine Dihydrochloride. After mixing, the assay mixture wasincubated at room temperature for 5 minutes, and MPO enzyme activitydetermined spectrophotometrically at 550 nanometers. Samples wereassayed in duplicate.

Inflammatory Mediator Secretion Studies

Four different leukocyte types or models that secrete specific granulecontents in response to phorbol ester induced activation of PKC wereused. Neutrophils were isolated from human blood and in vitro release ofMPO by these cells was assessed. Release of membrane-bound inflammatorymediators from commercially-available human leukocyte cell lines wasalso evaluated. The human promyelocytic cell line HL-60 clone 15 wasused to assess secretion of EPO (Fischkoff S A. Graded increase inprobability of eosinophilic differentiation of HL-60 promyelocyticleukemia cells induced by culture under alkaline conditions. Leuk Res1988; 12:679-686; Rosenberg H F, Ackerman S J, Tenen D G. Humaneosinophil cationic protein: molecular cloning of a cytotoxin andhelminthotoxin with ribonuclease activity. J Exp Med 1989; 170:163-176;Tiffany H L, Li F, Rosenberg H F. Hyperglycosylation of eosinophilribonucleases in a promyelocytic leukemia cell line and indifferentiated peripheral blood progenitor cells. J Leukoc Biol 1995;58:49-54; Badewa A P, Hudson C E, Heiman A S. Regulatory effects ofeotaxin, eotaxin-2, and eotaxin-3 on eosinophil degranulation andsuperoxide anion generation. Exp Biol Med 2002; 227:645-651). Themonocytic leukemia cell line U937 was used to assess secretion oflysozyme (Hoff T, Spencker T, Emmendoerffer A., Goppelt-Struebe M.Effects of glucocorticoids on the TPA-induced monocytic differentiation.J Leukoc Biol 1992; 52:173-182; Balboa M A, Saez Y, Balsinde J.Calcium-independent phospholipase A2 is required for lysozyme secretionin U937 promonocytes. J Immunol 2003; 170:5276-5280; Sundstrom C,Nilsson K. Establishment and characterization of a human histiocyticlymphoma cell line (U-937). Int J Cancer 1976; 17:565-577). Thelymphocyte natural killer cell line NK-92 used to assess release ofgranzyme (Gong J H., Maki G, Klingemann H G. Characterization of a humancell line (NK-92) with phenotypical and functional characteristics ofactivated natural killer cells. Leukemia 1994; 8:652-658; Maki G,Klingemann H G, Martinson J A, Tam Y K. Factors regulating the cytotoxicactivity of the human natural killer cell line, NK-92. J Hematother StemCell Res 2001; 10:369-383; Takayama H, Trenn G, Sitkovsky M V. A novelcytotoxic T lymphocyte activation assay. J Immunol Methods 1987;104:183-190). In all cases, the cells were preincubated with a range ofconcentrations of a synthetic peptide identical to the 24 amino acidMARCKS N-terminus (MANS-myristoylated N-terminal sequence peptide;MA-GAQFSKTAAKGEAAAERPGEAAVA wherein MA is myristoyl attached to theN-terminal amine of the peptide by an amide bond), or a missense controlpeptide (RNS: Random N-terminal sequence peptide;MA-GTAPAAEGAGAEVKRASAEAKQAF,) which consists of the same 24 amino acidsbut arranged in random order sequence which possesses less than 13%sequence identity to the MANS peptide sequence.

In each of the cell types, MANS, but not RNS, attenuates release ofinflammatory mediators in a concentration-dependent manner. A usefultime course of observation is 0.5-3.0 hrs. The results are consistentwith the N-terminal region of the MARCKS protein being involved inintracellular pathways leading to leukocyte degranulation.

Human Neutrophil Isolation—

These studies were approved by the NCSU human studies InstitutionalReview Board (IRB). Human neutrophils were isolated as previouslydescribed (see Takashi S, Okubo Y, Horie S. Contribution of CD54 tohuman eosinophil and neutrophil superoxide production. J Appl Physiol2001; 91:613-622) with slight modifications. Briefly, heparinized venousblood was obtained from normal healthy volunteers, diluted withRPMI-1640 (Cellgro; Mediatech, Inc., Herndon, Va.) at a ratio of 1:1,layered onto a Histopaque (density, 1.077 g/ml; Sigma-Aldrich Co., St.Louis, Mo.) and centrifuged at 400 g for 20 min at 4° C. The supernatantand mononuclear cells at the interface were carefully removed, anderythrocytes in the sediment were lysed in chilled distilled water.Isolated granulocytes were washed twice with Hanks' balanced saltssolution (HBSS) and resuspended in HBSS on ice. The neutrophils used forthe experiments were of >98% purity with <2% contamination byeosinophils, and the viability was >99% as determined by Trypan blue dyeexclusion.

Measurement of Released Neutrophil MPO Activity—

For measurement of MPO release, purified human neutrophils suspended inHBSS were aliquoted at 4×10⁶ cells/ml in 15 ml tubes and preincubatedwith either 50 or 100 μM of MANS or RNS peptide for 10 min at 37° C. Thecells then were stimulated with 100 nM phorbol 12-myristate 13-acetate(PMA) for up to 3 hrs. A control reference (PMA control reference) wasestablished using purified human neutrophils suspended in HBSS aliquotedat 4×10⁶ cells/ml in 15 ml tubes and stimulated with 100 nM phorbol12-myristate 13-acetate (PMA) in the absence of MANS or RNS peptide forthe same time periods. The reaction was terminated by placing the tubeson ice and centrifugation at 400 g for 5 min at 4° C.

MPO activity in the cell supernatant was assayed usingtetramethylbenzidine (TMB) based on a previously established technique(Abdel-Latif D, Steward M, Macdonald D L, Francis G A., Dinauer M C,Lacy P. Rac2 is critical for neutrophil primary granule exocytosis.Blood 2004; 104:832-839). Briefly, 100 μl of TMB substrate solution wasadded to 50 μl of cell supernatants or standard human MPO (EMDBiosciences, Inc., San Diego, Calif.) in a 96-well microplate followedby incubation at room temperature for 15 min. The reaction wasterminated by addition of 50 μl of 1M H₂SO₄ and absorbance was read at450 nm in a spectrophotometric microplate reader (VERSA max, MolecularDevices, Sunnyvale, Calif.).

Leukocyte Culture Studies.

Three types of human leukocyte cell lines, specifically thepromyelocytic cell line HL-60 clone 15, the monocytic cell line U937,and the lymphocyte natural killer cell line NK-92 were purchased fromAmerican Type Culture Collection (ATCC; Rockville, Md.). HL-60 clone 15cells (ATCC CRL-1964) were maintained in medium consisting of RPMI 1640with L-glutamine supplemented with 10% heat-inactivated fetal bovineserum (FBS; Gibco; Invitrogen Co., Carlsbad, Calif.), 50 IU/mlpenicillin, 50 μg/ml streptomycin, and 25 mM HEPES buffer, pH 7.8, at37° C. in an atmosphere containing 5% CO2. Final differentiation to aneosinophil-like phenotype was initiated by culturing cells at 5×10⁵cells/ml in the above medium containing 0.5 mM butyric acid(Sigma-Aldrich Co.) for 5 days as previously described (Tiffany H L, LiF, Rosenberg H F. Hyperglycosylation of eosinophil ribonucleases in apromyelocytic leukemia cell line and in differentiated peripheral bloodprogenitor cells. J Leukoc Biol 1995; 58:49-54; Tiffany H L, Alkhatib G,Combadiere C, Berger E A, Murphy P M. CC chemokine receptors 1 and 3 aredifferentially regulated by IL-5 during maturation of eosinophilic HL-60cells. J Immunol 1998; 160:1385-1392). U937 cells (ATCC CRL-1593.2) weregrown at 37° C. in an atmosphere of 5% CO₂ in complete medium consistingof RPMI 1640 with L-glutamine supplemented with 10% FBS, 50 IU/mlpenicillin, and 50 μg/ml streptomycin. NK-92 cells (ATCC CRL-2407) weremaintained in alpha-MEM medium (Sigma-Aldrich Co.) supplemented with 20%FBS, 100 U/ml of interleukin-2 (IL-2) (Chemicon International, Inc.,Temecula, Calif.), 5×10⁻⁵ M of 2-mercaptoethanol, 50 IU/ml penicillin,and 50 μg/ml streptomycin at 37° C. in an atmosphere containing 5% CO₂.Cell morphology was judged by assessment of Wright-Giemsa-stained cells.Viability of cells harvested for experiments was assessed by trypan blueexclusion and populations of cells with viability >95% were used.

Incubation of Cells for Degranulation Assays.

HL-60 clone 15, U937, and NK-92 cells were washed and resuspended at2.5×10⁶ cells/ml in phenol red-free RPMI-1640 (Cellgro; Mediatech, Inc.)for all degranulation assays. Aliquots of cells in 15 ml tubes werepreincubated with indicated concentrations of MANS or RNS peptide for 10min at 37° C. The cells then were stimulated with PMA for up to 2 hr. Acontrol reference (PMA control reference) was established for each celltype using HL-60 clone 15, U937, and NK-92 cells, respectively, whichwere washed and resuspended at 2.5×10⁶ cells/ml in phenol red-freeRPMI-1640 and stimulated with PMA but in the absence of MANS or RNSpeptide for the same time periods. The reaction was terminated byplacing tubes on ice and centrifuging cells at 400 g for 5 min at 4° C.

For measurements of released MPO from neutrophils and released lysozymefrom U937 cells, we were able to quantify secretion by using asstandards human MPO and egg white ovalbumin, respectively. For releasedEPO from HL-60 clone 15 cells and for released granzyme from NK-92cells, no standards were available to use for quantification. Hence,both released and intracellular (from lysed cells) levels of EPO andgranzyme were measured, and the released EPO and granzyme were expressedas a percentage of total (intracellular and released) for each. Tomeasure intracellular EPO in HL-60 clone 15 cells and intracellulargranzyme in NK-92 cells, appropriate aliquots of 0.1% triton X-100-lysedcells were taken for quantification of intracellular granule proteins asdescribed below. All treatments were expressed as percentage of controlto minimize variability between cultures.

Measurement of HL-60 EPO Release.

EPO activity released by HL-60 clone 15 cells was assayed using TMBaccording to a previously established technique (Lacy P, Mahmudi-Azer S,Bablitz B, Hagen S C, Velazquez J R, Man S F, Moqbel R. Rapidmobilization of intracellularly stored RANTES in response tointerferon-gamma in human eosinophils. Blood 1999; 94:23-32). Thus, 100μl of TMB substrate solution was added to 50=microliters) of sample in a96-well microplate and incubated at room temperature for 15 min(min=minutes). The reaction was terminated by addition of 50 μl of 1.0MH₂SO₄ and absorbance was read at 450 nm (nm=nanometers) in aspectrophotometric microplate reader. The amount of secreted EPO wasexpressed as percentage of total content, using the amount obtained inthe same number of triton X-100-lysed cells.

Measurement of Monocyte Lysozyme Secretion.

Lysozyme secreted by U937 cells was measured using a spectrophotometricassay as described previously (Balboa M A, Saez Y, Balsinde J.Calcium-independent phospholipase A2 is required for lysozyme secretionin U937 promonocytes. J Immunol 2003; 170:5276-5280) with slightmodification. Thus, 100 μl of sample was mixed with 100 μl of aMicrococcus lysodeikticus (Sigma-Aldrich Co.) suspension (0.3 mg/ml in0.1 M sodium phosphate buffer, pH 7.0) in a 96-well microplate. Thedecrease in absorbance at 450 nm was measured at room temperature. Acalibration curve was constructed using chicken egg white lysozyme (EMDBiosciences, Inc.) as a standard.

Measurement of NK Cell Granzyme Secretion.

Granzyme secreted from NK-92 cells was assayed by measuring hydrolysisof Na-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) essentially asdescribed previously (Takayama H, Trenn G, Sitkovsky M V. A novelcytotoxic T lymphocyte activation assay. J Immunol Methods 1987;104:183-190). 50 μl of supernatant was transferred to a 96-well plate,and 150 μl of BLT solution (0.2 mM BLT; EMD Biosciences, Inc., and 0.22mM DTNB; Sigma-Aldrich Co.) (mM=millimolar) in phosphate-buffered saline(PBS, pH 7.2) was added to the supernatant. Absorbance at 410 nm wasread after incubation for 30 min at room temperature. Results wereexpressed as percentage of total cellular enzyme content, using theamount obtained in the same number of triton X-100-lysed cells.

Statistical Analysis.

Statistical significance of the differences between various treatmentgroups was assessed with one-way ANOVA. P values of <0.05 were taken assignificant.

Neutrophil MPO Release

It was found that 100 nM PMA (as a stimulator of inflammatory mediatorrelease) increased human neutrophil MPO release by approximatelythreefold versus control level at 30 min in the PMA control reference,the release of MPO increasing to approximately 5-6 fold after 3 hrs. At30 minutes, relative to the control MPO activity as 100% absent PMA andabsent PMA plus MANS or RNS, MPO activity of the PMA control referencewas about 275%, PMA plus 50 μM MANS was about 275%, and 100 μM MANS wasabout 305%. Thus, the MANS peptide had no detected effect at 30 min.However, by 1 hr the higher concentration of MANS (100 μM) had asignificant inhibitory effect (measured at about 260% of control) orabout 25% reduction in MPO release versus the PMA control referencelevel (which was measured at about 340% of control). The 50 μM MANSsample measured about 290% of control or about 15% reduction relative tothe PMA control reference. By 2 hrs and persisting at 3 hrs, the MANSpeptide significantly attenuated MPO activity in aconcentration-dependent manner. At 2 hours, the PMA control referenceMPO activity was about 540% of control, the 50 μM MANS (measuring about375% of control) caused an approximately 30% reduction of MPO releaseversus the PMA control reference; and 100 μM MANS (measuring about 295%of control) caused an approximately 45% reduction of MPO release versusthe PMA control reference. At 3 hours, the PMA control reference MPOactivity was about 560% of control, 50 μM MANS (measuring about 375% ofcontrol) caused an approximately 33% reduction of MPO release versus thePMA control reference; 100 μM MANS (measuring about 320% of control)caused an approximately 40% reduction of MPO release versus the PMAcontrol reference. The RNS peptide did not affect PMA-induced MPOrelease at any of the time points or concentrations tested.

HL-60 EPO Release

EPO activity in the supernatant of HL-60 clone 15 cells wassignificantly enhanced at 1 and 2 hrs after PMA stimulation. At 1 hour,relative to EPO activity of the control as 100%, the PMA controlreference measured at about 110%; the sample containing 10 μM MANSmeasured at about 95% to give about 15% reduction in EPO activityrelative to the PMA control reference; the sample containing 50 μM MANSmeasured at about 78% to give about 30% reduction in EPO activityrelative to the PMA control reference; and the sample containing 100 μMMANS measured at about 65% to give about 40% reduction in EPO activityrelative to the PMA control reference. At 2 hour, relative to EPOactivity of the control as 100%, the PMA control reference measured atabout 145%; the sample containing 10 μM MANS measured at about 130% togive about 10% reduction in EPO activity relative to the PMA controlreference; the sample containing 50 μM MANS measured at about 70% togive about 50% reduction in EPO activity relative to the PMA controlreference; and the sample containing 100 μM MANS measured at about 72%to give about 50% reduction in EPO activity relative to the PMA controlreference. Thus, at both 1 and 2 hrs, MANS at 50 or 100 μM significantlyattenuated EPO release. The RNS peptide did not affect PMA-enhanced EPOrelease at any of the time points or concentrations tested.

U937 Lysozyme Release

Lysozyme secretion by U937 cells was increased by PMA stimulation by 1hr after incubation, and increased even more at 2 hrs. At 1 hour,relative to lysozyme secretion by U937 cells of the control as 100%, thePMA control reference measured at about 210%; the sample containing 10μM MANS measured at about 170% to give about 20% reduction in lysozymesecretion by U937 cells relative to the PMA control reference; thesample containing 50 μM MANS measured at about 170% to give about 20%reduction in lysozyme secretion by U937 cells relative to the PMAcontrol reference; and the sample containing 100 μM MANS measured atabout 115% to give about 45% reduction in lysozyme secretion by U937cells relative to the PMA control reference. At 2 hour, relative tolysozyme secretion by U937 cells of the control as 100%, the PMA controlreference measured at about 240%; the sample containing 10 μM MANSmeasured at about 195% to give about 20% reduction in lysozyme secretionby U937 cells relative to the PMA control reference; the samplecontaining 50 μM MANS measured at about 185% to give about 25% reductionin lysozyme secretion by U937 cells relative to the PMA controlreference; and the sample containing 100 μM MANS measured at about 140%to give about 40% reduction in lysozyme secretion by U937 cells relativeto the PMA control reference. Thus, lysozyme secretion was significantlyattenuated at both 1 and 2 hours post-stimulation by 100 μM of MANS butnot as much by 50 or 10 μM of MANS. The RNS peptide did not affectPMA-enhanced lysozyme secretion at any of the time points orconcentrations tested.

NK Cell Granzyme Release

At 1 hour, relative to granzyme secretion by NK-92 cells of the controlas 100%, the PMA control reference measured at about 125%; the samplecontaining 10 μM MANS measured at about 115% to give about 10% reductionin granzyme secretion by NK-92 cells relative to the PMA controlreference; and the sample containing 100 μM MANS measured at about 85%relative to the PMA control reference to give about 30% reduction ingranzyme secretion by NK-92 cells relative to the PMA control reference.At 2 hour, relative to granzyme secretion by NK-92 cells of the controlas 100%, the PMA control reference measured at about 220%; the samplecontaining 10 μM MANS measured at about 200% to give about 10% reductionin granzyme secretion by NK-92 cells relative to the PMA controlreference; and the sample containing 100 μM MANS measured at about 80%to give about 60% reduction granzyme secretion by NK-92 cells relativeto the PMA control reference. Thus, granzyme secretion by NK-92 cellswas not significantly increased by PMA at 1 hr, but increased overtwo-fold at 2 hours. 100 μM of MANS, but not 10 μM of MANS, attenuatedgranzyme secretion at 1 and 2 hrs after incubation. The RNS peptide didnot affect PMA-enhanced granzyme secretion at any of the time points orconcentrations tested.

Cytotoxicity

None of the treatments generated a toxic response in the cells, asassessed by LDH retention/release (data not shown) (see also Park J-A,He F, Martin L D, Li Y, Adler K B. Human neutrophil elastase induceshypersecretion of mucin from human bronchial epithelial cells in vitrovia a PKCδ-mediated mechanism. Am J Pathol 2005; 167:651-661).

The foregoing examples are illustrative of the present invention and arenot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

We claim:
 1. A method of inhibiting release of an inflammatory mediatorassociated with a chronic inflammatory disease in a subject comprisingcontacting an inflammatory cell in the subject suffering from a chronicinflammatory disease, which cell comprises the inflammatory mediatorcontained within a granule inside the cell, with at least one activemyristoylated N-terminal fragment of a MANS peptide (SEQ ID NO:1),wherein said fragment comprises at least six contiguous amino acids ofsaid MANS peptide, in an effective amount to reduce the MARCKS-relatedrelease of the inflammatory mediator from the inflammatory cell.
 2. Themethod of claim 1, wherein said inflammatory mediator is selected fromthe group consisting of myeloperoxidase (MPO), eosinophil peroxidase(EPO), major basic protein (MBP), lysozyme, granzyme, histamine,proteoglycan, protease, a chemotactic factor, cytokine, a metabolite ofarachidonic acid, defensin, bactericidal permeability-increasing protein(BPI), elastase, cathepsin G, cathepsin B, cathepsin D,beta-D-glucuronidase, alpha-mannosidase, phospholipase A₂,chondroitin-4-sulphate, proteinase 3, lactoferrin, collagenase,complement activator, complement receptor,N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, lamininreceptor, cytochrome b₅₅₈, monocyte-chemotactic factor, histaminase,vitamin B12 binding protein, gelatinase, plasminogen activator, and acombination thereof.
 3. The method according to claim 1, wherein saidactive fragment can be selected from the group consisting ofN-myristoyl-GAQFSKTAAKGEAAAERPGEAAV (SEQ ID NO: 20);N-myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO: 3);N-myristoyl-GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 4);N-myristoyl-GAQFSKTAAKGEAAAERPGE (SEQ ID NO: 5);N-myristoyl-GAQFSKTAAKGEAAAERPG (SEQ ID NO: 6);N-myristoyl-GAQFSKTAAKGEAAAERP (SEQ ID NO: 7);N-myristoyl-GAQFSKTAAKGEAAAER (SEQ ID NO: 8);N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID NO: 9); N-myristoyl-GAQFSKTAAKGEAAA(SEQ ID NO: 10); N-myristoyl-GAQFSKTAAKGEAA (SEQ ID NO: 11);N-myristoyl-GAQFSKTAAKGEA (SEQ ID NO: 12); N-myristoyl-GAQFSKTAAKGE (SEQID NO: 13); N-myristoyl-GAQFSKTAAKG (SEQ ID NO: 14);N-myristoyl-GAQFSKTAAK (SEQ ID NO: 15); N-myristoyl-GAQFSKTAA (SEQ IDNO: 16); N-myristoyl-GAQFSKTA (SEQ ID NO: 17); N-myristoyl-GAQFSKT (SEQID NO: 18); and N-myristoyl-GAQFSK (SEQ ID NO: 19).
 4. The methodaccording to claim 1, wherein said inflammatory cell is a leukocyte. 5.The method according to claim 1, wherein said inflammatory cell is agranulocyte.
 6. The method according to claim 1, wherein saidinflammatory cell is selected from the group consisting of a neutrophil,a basophil, an eosinophil and a combination thereof.
 7. The methodaccording to claim 1, wherein said inflammatory cell is a monocyte ormacrophage.
 8. The method according to claim 1, wherein saidinflammatory mediator is selected from the group consisting ofmyeloperoxidase (MPO), eosinophil peroxidase (EPO), lysozyme, granzymeand a combination thereof.
 9. The method according to claim 1, whereinsaid effective amount comprises a degranulation-inhibiting amount of anactive fragment of MANS peptide that reduces the amount of aninflammatory mediator released from said inflammatory cell from about 1%to about 99% as compared to the amount released from said inflammatorycell in the absence of said active fragment.
 10. The method according toclaim 1, wherein said effective amount comprises adegranulation-inhibiting amount of an active fragment of MANS peptidethat reduces the amount of an inflammatory mediator released from saidinflammatory cell from between about 5-50% to about 99% as compared tothe amount released from said inflammatory cell in the absence of saidactive fragment.
 11. The method of claim 1, wherein the chronicinflammatory disease is selected from the group consisting ofinsulin-dependent diabetes mellitus, multiple sclerosis, Gullian-Barresyndrome, graft versus host disease, and systemic lupus erythematosus.12. A method of inhibiting release of an inflammatory mediatorassociated with a chronic inflammatory disease in a subject comprising:administration to a tissue and/or fluid of said subject suffering from achronic inflammatory disease, wherein said tissue and/or said fluidcomprises at least one inflammatory cell comprising at least oneinflammatory mediator contained within a granule inside the cell, atherapeutically effective amount of a pharmaceutical compositioncomprising at least one active myristoylated N-terminal fragment of aMANS peptide (SEQ ID NO:1), wherein said fragment comprises at least sixcontiguous amino acids of said MANS peptide, in a therapeuticallyeffective amount to reduce the MARCKS-related release of said at leastone inflammatory mediator from said inflammatory cell.
 13. The method ofclaim 12, wherein said inflammatory mediator is selected from the groupconsisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO), majorbasic protein (MBP), lysozyme, granzyme, histamine, proteoglycan,protease, a chemotactic factor, cytokine, a metabolite of arachidonicacid, defensin, bactericidal permeability-increasing protein (BPI),elastase, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase,alpha-mannosidase, phospholipase A₂, chondroitin-4-sulphate, proteinase3, lactoferrin, collagenase, complement activator, complement receptor,N-formylmethionyl-leucyl-phenylalanine (FMLP) receptor, lamininreceptor, cytochrome b₅₅₈, monocyte-chemotactic factor, histaminase,vitamin B12 binding protein, gelatinase, plasminogen activator, and acombination thereof.
 14. The method according to claim 12, wherein saidreducing the MARCKS-related release of said inflammatory mediatorcomprises blocking or inhibiting the mechanism that releases saidinflammatory mediator from said inflammatory cell.
 15. The methodaccording to claim 12, wherein said active fragment can be selected fromthe group consisting of N-myristoyl-GAQFSKTAAKGEAAAERPGEAAV (SEQ ID NO:20); N-myristoyl-GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO: 3);N-myristoyl-GAQFSKTAAKGEAAAERPGEA (SEQ ID NO: 4);N-myristoyl-GAQFSKTAAKGEAAAERPGE (SEQ ID NO: 5);N-myristoyl-GAQFSKTAAKGEAAAERPG (SEQ ID NO: 6);N-myristoyl-GAQFSKTAAKGEAAAERP (SEQ ID NO: 7);N-myristoyl-GAQFSKTAAKGEAAAER (SEQ ID NO: 8);N-myristoyl-GAQFSKTAAKGEAAAE (SEQ ID NO: 9); N-myristoyl-GAQFSKTAAKGEAAA(SEQ ID NO: 10); N-myristoyl-GAQFSKTAAKGEAA (SEQ ID NO: 11);N-myristoyl-GAQFSKTAAKGEA (SEQ ID NO: 12); N-myristoyl-GAQFSKTAAKGE (SEQID NO: 13); N-myristoyl-GAQFSKTAAKG (SEQ ID NO: 14);N-myristoyl-GAQFSKTAAK (SEQ ID NO: 15); N-myristoyl-GAQFSKTAA (SEQ IDNO: 16); N-myristoyl-GAQFSKTA (SEQ ID NO: 17); N-myristoyl-GAQFSKT (SEQID NO: 18); and N-myristoyl-GAQFSK (SEQ ID NO: 19).
 16. The methodaccording to claim 12, wherein said inflammatory cell is a leukocyte.17. The method according to claim 12, wherein said inflammatory cell isa granulocyte.
 18. The method according to claim 12, wherein saidinflammatory cell is selected from the group consisting of a neutrophil,a basophil, an eosinophil and a combination thereof.
 19. The methodaccording to claim 12, wherein said inflammatory cell is a monocyte ormacrophage.
 20. The method according to claim 12, wherein saidinflammatory mediator is selected from the group consisting ofmyeloperoxidase (MPO), eosinophil peroxidase (EPO), lysozyme, granzymeand a combination thereof.
 21. The method according to claim 12, whereinsaid effective amount comprises a degranulation-inhibiting amount of anactive fragment of MANS peptide that reduces the amount of aninflammatory mediator released from said inflammatory cell from about 1%to about 99% as compared to the amount released from said inflammatorycell in the absence of said active fragment.
 22. The method according toclaim 12, wherein said effective amount comprises adegranulation-inhibiting amount of an active fragment of MANS peptidethat reduces the amount of an inflammatory mediator released from saidinflammatory cell from about 5 to 50% to about 99% as compared to theamount released from said inflammatory cell in the absence of saidactive fragment.
 23. The method according to claim 12, wherein saidchronic inflammatory disease is selected from the group consisting ofinsulin-dependent diabetes mellitus, multiple sclerosis, Gullian-Barresyndrome, graft versus host disease, and systemic lupus erythematosus.24. The method according to claim 12, wherein said subject is a mammal.25. The method according to claim 12, wherein said mammal is selectedfrom the group consisting of a human, a canine, an equine and a feline.26. The method according to claim 12, wherein said administration isselected from the group consisting of topical administration, parenteraladministration, rectal administration, pulmonary administration, nasaladministration, and oral administration.
 27. The method according toclaim 26, wherein said pulmonary administration is selected from thegroup of aerosol, dry powder inhaler, metered dose inhaler, andnebulizer.
 28. The method according to claim 12, further comprisingadministration to said subject of a second molecule selected from thegroup consisting of an antibiotic, an antiviral compound, anantiparasitic compound, an anti-inflammatory compound, and animmunosuppressant.